Antigenic Properties of Virion Polypeptides

Transcription

1 JOURNAL OF VIROLOGY, Feb. 1983, p X/83/ $02.00/0 Copyright C) 1983, American Society for Microbiology Vol. 45, No. 2 Porcine Parvovirus: Virus Purification and Structural and Antigenic Properties of Virion Polypeptides THOMAS W. MOLITOR,1 H. S. JOO,1 AND MARC S. COLLETT2* Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108,1 and Department of Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota Received 18 October 1982/Accepted 10 November 1982 Porcine parvovirus (PPV) was extensively purified from infected swine fetal homogenates by CaC12 precipitation followed by CsCl density centrifugation. Two species of particles possessing PPV-specific hemagglutinating activity were observed banding at densities of 1.39 and 1.30 g/ml, representing full and empty 20-nm virion particles, respectively. Both classes of particles contained three major polypeptides, A, B, and C, with respective molecular weights of 83,000, 64,000, and 60,000. The amount of polypeptide A was similar in both species (approximately 10%); however, the B protein was most abundant in the 1.30-g/ml particles, whereas the C protein was the major polypeptide found in the 1.39-g/ml particles. Antisera generated to each sodium dodecyl sulfate-polyacrylamide gelpurified virion structural protein had reactivities qualitatively similar to those of conventional antisera raised against intact PPV in a variety of standard serological assays. The antisera generated against the individual sodium dodecyl sulfatedenatured PPV polypeptides were able to react with native, intact PPV virions and were capable of neutralizing virus infectivity. Porcine parvovirus (PPV) is a ubiquitous infectious agent of pigs. The etiological role of this virus in reproductive failure in swine is well established (13, 20, 21). Most often, disease caused by PPV is manifested as fetal death and mummification, although infertility, abortion, stillbirth, and neonatal death may also be consequences of in utero PPV infection (13, 21). Humoral immunity, either as a consequence of natural exposure or from vaccination, will often prevent viremia in pregnant sows and thus prevent transplacental virus infection and subsequent fetal death (15, 20). PPV, like other parvoviruses, is a small, nonenveloped virus containing single-stranded DNA (23). PPV appears to be a nondefective or autonomously replicating parvovirus, similar in this respect to the more extensively studied parvoviruses such as the minute virus of mice and the Kilham rat virus, and in contrast to the defective adeno-associated viruses (35). Very little is known of the molecular features of PPV. We began our study of PPV by developing a scheme for obtaining the virus in highly purified form from infected swine fetal tissue and then proceeded with studies on the virion protein composition. We report here our work on the isolation and purification of PPV from infected fetuses and the initial characterization of various features of the three major virion 842 polypeptides. We prepared antisera to each purified viral polypeptide and compared the reactivities of these antisera in a variety of tests. We found that the three virion proteins of PPV were both structurally and antigenically quite similar. Finally, the denatured, gel-purified virion proteins were able to elicit virus-neutralizing antibody. MATERIALS AND METHODS Virus propagation in infected animals. Pregnant sows, at approximately 35 to 40 days of gestation, were purchased from a local swine producer. The serological status of these sows was not important because no transplacental immunity has been observed in swine (30). At 40 to 50 days, sows were laparotimized, and fetuses were infected via amniotic fluid inoculation with PPV (0.2 ml; 1,024 hemagglutinating units per 50,ul; % tissue culture infective doses) as previously described (25). The virus used for inoculation was PPV strain NADL-8, originally obtained from the National Animal Disease Laboratory, Ames, Iowa. Ten to 15 days later, the sows were taken to slaughter; the entire uterus of each was collected and transported back to the laboratory. Pooled internal tissues from each fetus (lung, kidneys, liver, heart, spleen, and intestines) were collected and minced in 50 mm Tris-hydrochloride-25 mm EDTA buffer, ph 8.7 (TE buffer). After overnight freezing, minced tissue fluids from each fetus were tested by hemagglutination (HA) of guinea pig erythrocytes for presence of virus (see below). Tissues from fetuses that were highly HA

2 VOL. 45, 1983 PPV: PURIFICATION AND ANTIGENIC PROPERTIES 843 Infected fetuses (viscera) 20% homogenate/sonic extract in TE buffer HA assay individual fetuses HA positive HA negative: discard -12,000 x g, 30 min Supernatant Fractions - Discontinuous Supernatant Resuspend in TE buffer Clear Discard pellet Adjust to 25 mm CaCl,, 4 C, 30 min 12,000 x g, 20 min CsCl gradient 120,000 x g, 24 h Discard supernatant K Determine fraction density Assay for viral antigen by HA Pool virus peak (1.39 g/ml), dialyze against TE buffer Repeat discontinuous CsCl gradient Pool virus peak (1.39 g/ml), dialyze against TE buffer Purified PPV FIG. 1. Scheme for purification of PPV. positive (>1:i,024) were pooled, diluted to a 20% (wt/ vol) suspension in additional TE buffer, and further homogenized in a Waring blender. The suspension was frozen and thawed twice and was sonicated at 4 C for three 1-min intervals before virus purification. Virus purification. The procedure for the purification of PPV from mummified fetuses was adapted from the procedure of Tattersall et al. (33) used for minute virus of mice (Fig. 1). All manipulations were carried out at 4 C. Sonicated homogenates of HA-positive fetal viscera were cleared of cellular debris by centrifugation at 12,000 x g for 30 min. To the resultant supernatant was added CaCl2 dropwise to a final concentration of 25 mm, and the virus was allowed to precipitate for 30 min. The precipitate was collected by centrifugation at 12,000 x g for 20 min and was suspended by gentle sonic treatment in TE buffer. Any insoluble material was removed by brief centrifugation, and the cleared supernatant was adjusted to a final CsCl density of 1.32 g/ml in TE buffer. This solution was layered onto an equal volume of a CsCI solution in TE buffer having a density of 1.40 g/ml. Centrifugation was performed at 120,000 x g for 18 h at 4 C (Beckman SW41 rotor). Gradients were fractionated (approximately 25 to 30 fractions), the refractive index of each fraction was determined, and the presence of viral antigen was assayed by HA. Fractions containing hemagglutinating activity were pooled and subjected to a second cycle of discontinuous CsCl centrifugations as outlined in the legend to Fig. 2. The pooled HA-positive fractions from the second CsCl gradients were then dialyzed against TE buffer. Radioiodination of viral proteins. Viral proteins were labeled with 1251, using the chloramine T method of Hunter (8). Purified virus (50 to 100,ug) in TE buffer (50,il) was disrupted by boiling in 1% sodium dodecyl

3 844 MOLITOR, JOO, AND COLLETT sulfate (SDS) for 3 min. After cooling to room temperature, 100,Ci of '25I (carrier free; New England Nuclear Corp.) was added, followed by 50,ug of chloramine T. After 2 min at room temperature, the reaction mixture was applied to a Sephadex G-50 column (1 by 12 cm) equilibrated with 0.01 M sodium phosphate (ph 6.8). The excluded peak of radioactivity was collected and pooled. The specific activity of the radiolabeled proteins was approximately 5 x 106 cpm/,ug. Viral polypeptide purification. Viral structural proteins, either '25l radiolabeled or unlabeled, were boiled in SDS-containing sample buffer for 2 min and then were subjected to electrophoresis in SDS-containing 10% polyacrylamide gels (17). Proteins were localized either by autoradiography or by Coomassie brilliant blue staining of end sections of the gels. The individual protein bands were excised, and the protein-containing gel pieces were subjected to another cycle of electrophoresis on a second SDS-containing polyacrylamide gel. After protein localization in the second gel, the excised gel pieces were either used directly for one-dimensional partial proteolytic peptide mapping (see below) or used for the extraction of the protein contained within. Isolation of viral proteins from polyacrylamide gel pieces was either by repeated elution in 0.05 M NH4HCO3-0.1% SDS at 37 C or by isotachophoresis in Sephadex G-50 columns (1). The leading buffer was 50 mm Tris-hydrochloride (ph 6.8), and the terminating buffer was 50 mm Tris-glycine (ph 8.8). Bromophenol blue was used as a tracking dye for the leading edge of protein. The dye-containing Sephadex was removed from the columns and washed with water to recover the protein. Both procedures resulted in recoveries of protein of 65 to 90%. To prepare the individual virion polypeptides for use in animal immunizations, proteins were twice gel purified and eluted from the polyacrylamide gel pieces in 0.05 M NH4HCO3-0.1% SDS as described above. The eluates were lyophilized, suspended in 1 to 2 ml of TE buffer, and extensively dialyzed over a period of several days against buffer containing 10 mm potassium phosphate (ph 6.8), 40 mm NaCl, 1 mm EDTA, 1 mm 2- mercaptoethanol, and 50% glycerol. The method of Lowry et al. (18) was employed to determine protein concentrations, using bovine serum albumin (BSA) as a standard. Peptide mapping. The one-dimensional partial proteolysis mapping procedure of Cleveland et al. was employed as originally described (4). '25I-labeled PPV proteins, purified by two cycles of SDS-polyacryl- J. VIROL. amide gel electrophoresis, were subjected to electrophoresis in SDS-containing 15% polyacrylamide gels in the presence of either Staphylococcus aureus V8 protease (Miles Laboratories, Inc.), elastase (Worthington Diagnostics), or chymotrypsin (Worthington). Preparation of antisera. Normal porcine sera were collected from adult pigs that were free of PPVspecific antibodies as determined by hemagglutination inhibition (HAI; see below) and by immunoprecipitation of radiolabeled PPV-infected cultured cell lysates (data not shown). Adult pig sera containing antibodies to PPV, as determined by HAI, were collected from sows or gilts naturally exposed to PPV. Fetal pig sera containing antibodies to PPV were prepared by the amniotic injection of 70- to 85-day-old fetuses with 0.2 ml of PPV NADL-8 (1,024 hemagglutination units per 50,u1). Fetuses were collected 21 days later and bled. Rabbit antisera against intact PPV and gel-purified viral polypeptides were prepared in the following manner. Five-pound (2,268-g) New Zealand white rabbits were bled by the ear veins to obtain preimmune (normal) rabbit sera. These sera were demonstrated to lack antibodies specific for PPV antigens by HAI and by immunoprecipitation of radiolabeled PPV-infected cultured cell lysates; they were therefore used for subsequent immunizations. Rabbits were injected subcutaneously at multiple sites along the back with either 50,ug of intact, CsCl-purified, full virus particles or 50,ug of gel-purified polypeptide dissolved in 1 ml of TE buffer that had been emulsified in an equal volume of complete Freund adjuvant. One rabbit was used for each antigen preparation. Rabbits receiving purified viral polypeptides were injected at 3 weeks and 7 weeks after the initial immunization with 50 jig of the respective gel-purified protein in incomplete Freund adjuvant. The rabbit that received intact virus was boosted once at 3 weeks with 50 jig of virus. All rabbits were maintained in individual cages. Rabbits injected with live virus did not show demonstrable virus shedding, and other normal rabbits maintained in the same animal room remained negative for PPV antibodies throughout the course of this experiment. Table 1 summarizes the various antisera used in these studies. HA and HAI. PPV antigen was detected by the hemagglutination of guinea pig erythrocytes as previously described (14). Antibody titers to PPV were measured by an HAl test (14). Briefly, heat-inactivated sera were first absorbed with 25% kaolin in boratesaline solution (ph 9.0) for 20 min and centrifuged. TABLE 1. Various antisera used in the present study Antiserum Abbrevia- Method of production/source tion Normal pig serum NPS HAI-negative adult animal Adult pig appv P a PPV Naturally exposed field animal Fetal pig otppv F ox PPV Experimental virus infection by amniotic injection of pregnant sow Normal rabbit serum NRS Pre-immunization sera of rabbits used below Rabbit a intact PPV R a PPV Immunization with CsCl-purified intact virion particles Rabbit ot A polypeptide R a A Immunization with gel-purified A polypeptide; 10-week post-immunization serum Rabbit a B polypeptide R a B As above with B polypeptide Rabbit a C polypeptide R a C As above with C polypeptide

4 VOL. 45, 1983 This was followed by incubation for 60 min with 50% guinea pig erythrocytes and centrifugation (500 x g for 10 min). This procedure removed natural nonspecific hemagglutinins. Sera were then serially diluted with phosphate-buffered saline (PBS) in microtiter plates. A standard amount of cell culture-propagated PPV (8 hemagglutination units per 50.d) was added to all wells and allowed to incubate for 3 h at 22 C or overnight at 4 C. Then a 0.6% suspension of guinea pig erythrocytes was added to all wells and incubated at 22 C for 2 h. The endpoint is expressed as the reciprocal of the highest dilution resulting in 50% inhibition of HA Ȯther serological assays. An indirect fluorescentantibody (IFA) test, previously described by Bommeli et al. (3), was used to compare the reactivities of various antisera with infected cells in culture. Briefly. PPV-infected ST (swine testicle) cells grown on Lab- Tek slides were treated with acetone and air dried. Various antisera, diluted 1:8 in PBS, were incubated with the monolayers at 37 C for 60 min, after which they were washed and further incubated with 0.1 ml of a 1:50 dilution of a 1-mg/ml solution of protein A- fluorescein isothiocyanate conjugate (Pharmacia Fine Chemicals. Inc.). Controls consisted of known PPVpositive and -negative sera, uninfected ST cells, and pseudorabies virus-infected cells. Agar gel immunodiffusion (AGID) was performed as previously described (16) to test for the presence of precipitating antibodies. Protein transfer to nitrocellulose and reaction with antisera. PPV proteins were electrophoretically transferred from SDS-polyacrylamide gels to nitrocellulose paper (Schleicher & Schuell Co.) by the procedure of Bittner et al. (2). After transfer, gels were stained with Coomassie blue to estimate the efficiency of transfer. Routinely, 70 to 95% of the proteins were transferred to the nitrocellulose sheets. The procedure of Symington et al. (31) was employed to immunologically detect proteins. Briefly, the sheets containing the transferred proteins were placed in a solution of 4% BSA-0.01% sodium azide for 18 h at 22'C. Purified immunoglobulin G (100 to 200 p.g; obtained by ammonium sulfate precipitation) from various antisera was added. and incubation was continued with gentle shaking for 16 to 24 h at 22 C. Sheets were then washed five or six times (5 min per wash) with BSA-sodium azide solution. Protein A (Pharmacia), radioiodinated by the procedure of Hunter (8) in BSA-sodium azide solution (107 cpm; 8 x 106 cpm/[.g). was added, and the sheets were gently shaken at 22 C for 3 to 4 h. The sheets were again washed five or six times (10 min per wash) with BSA-sodium azide solution, air dried, and exposed to X-ray film. Electron microscopy. CsCl-purified virions, extensively dialyzed against TE buffer, were analyzed by negative staining with 4% phosphotungstic acid, using standard procedures (7). Grids were examined in a Zeiss 10 transmission electron microscope at a plate magnification of 40,000 with an 80-kV beam. PPV: PURIFICATION AND ANTIGENIC PROPERTIES 845 The ability of the various antisera to form lattices with intact virion particles was tested by an immunoelectron microscopy procedure (7). Various sera (diluted 1:10 in TE buffer) were mixed with equal volumes of purified PPV in TE buffer and incubated overnight at 4 C. The mixtures were then diluted 1:100 with water and centrifuged at 30,000 x g for 30 min to pellet the immune complexes. The pellets were resuspended in a small volume of TE buffer, negatively stained, and analyzed in the electron microscope as described above. Virus infectivity neutralization. Standard virus infectivity neutralization assays were performed in triplicate. Sera (1:2 or 1:10 dilutions in PBS, total volume 0.3 ml) were mixed and incubated with 0.3 ml of virus (8 HA units per 50 [LI) for 60 min at 22 C. A 200-,ul volume of this mixture was then added to Leighton tubes containing recently trypsinized ST cells previously washed with PBS. Controls included known positive sera. known negative sera. no serum, and no virus. The inocula were allowed to absorb for 90 to 120 min. after which the cultures were washed with PBS and fresh growth medium was added. Four days after inoculation, the Leighton slips were removed, treated with acetone, and air dried. The slips were then stained directly with fluorescent-antibody conjugate. washed, and observed under a fluorescent microscope. The fluorescent-antibody conjugate consisted of fluorescent isothiocyanate conjugated to porcine anti-ppv antibodies produced in gnotobiotic pigs (21). Virus infection of cells was considered positive when multiple foci of fluorescently labeled cells were apparent on any given Leighton slip. RESULTS Purification of PPV from infected fetuses. The purification procedure for PPV from infected porcine fetuses is outlined in Fig. 1. As found for minute virus of mice (33) and H-1 virus (24), PPV is significantly purified from cellular material at high ph and low ionic strength in the presence of EDTA and is greatly enriched for by CaCl. precipitation. CaCl precipitation of the virus was found to be essential for obtaining highly purified preparations and was far superior to other methods of virus concentration (e.g., 12% polyethylene glycol or 30% ammonium sulfate). The CaCl-precipitated virus was suspended in the high-ph buffer in the presence of 25 mm EDTA, and the soluble virus-containing material was then subjected to discontinuous CsCl density centrifugation (Fig. 2). As detected by HA, viral antigen appeared in two major, well-resolved peaks at buoyant densities of 1.39 and 1.30 g/ml (Fig. 2A). These two peaks were routinely seen in different fetal preparations and usually contained equal amounts of hemagglutinating activity. To further enrich for these two viral antigen-containing species, we subjected the pooled fractions of each density peak to a second cycle of discontinuous CsCl centrifugation. The 1.39-g/ml density particles were run on a density gradient identical to the first gradient (Fig. 2B), and the lighter 1.30-g/ml particles were centrifuged in a slightly less dense gradient (Fig. 2C). In each case, a single HA peak of viral antigen was observed, banding at the same density as found in the first density gradient run. The HA-positive fractions of both the and the 1.30-g/ml particles were individually pooled and dialyzed against TE buffer.

5 846 MOLITOR, JOO, AND COLLETT The purity of these two viral antigen-containing fractions was assessed by negative-contrast electron microscopy (Fig. 3A) of samples and Coomassie brilliant blue staining in SDS-containing 10% polyacrylamide gels electrophoresed (Fig. 3C). Particles banding at a density of 1.39 g/ml had a diameter of approximately 20 nm. They excluded the phosphotungstic acid stain (Fig. 3A) and therefore appeared as characteristic "full" (DNA-containing) parvovirus virions. Particles of similar size were found in the 1.30-g/ml density band together with varying amounts of debris (Fig. 3B). However, these particles appeared to largely take up the negative stain; therefore, they most likely represent "empty" (DNA-lacking) virion capsids. These results appear to be similar to those obtained from other, more thoroughly studied parvovirus systems (35). We have not attempted to identify viral particles that may be present at intermediate densities or that may represent various immature or partial genome-containing virions. The polypeptide composition of the material present in the and 1.30-g/ml density bands was analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 3C). Both preparations appeared to contain three predominant protein bands with molecular weights of 83,000, 64,000, and 60,000. The three proteins of the "full" particles appeared to be highly enriched, whereas minor, presumably contaminating protein species were present in the "empty" particle preparations. The presence of three polypeptides of the observed molecular weights in highly purified virus preparations is very characteristic of parvoviruses (5, 10, 11, 26-28, 33), and we therefore conclude that these three polypeptides are the structural proteins of PPV. The structural proteins of parvoviruses have previously been termed A, B, and C in descending order of molecular weight (32), although VP1, VP2, and VP3 have also been used (19, 24). The relative distribution of the three PPV structural proteins should be noted. In the g/ml particles, the 60-kilodalton (kd) protein (C or VP3) appears to be most abundant, whereas in the 1.30-g/ml particles, the 64-kd protein (B or VP2) is present in the greatest amounts (Fig. 3C). Although this difference in the B and C proteins between full and empty virion particles exists, the amount of the 83-kd protein (A or VP1) appears to remain constant (approximately 10% of the total). This observation has been made with several independent PPV preparations and has also been observed in other parvovirus systems (5, 24). Structural relatedness of PPV polypeptides. Previous studies of parvovirus structural proteins have indicated that the A, B, and C polypeptides are very closely related as determined 4 (0 z I J. VIROL. FRACTION NUMBER FIG. 2. CsCl density centrifugation of PPV. PPV was precipitated with CaC12 from virus-containing homogenates of infected swine fetuses, suspended in TE buffer, and prepared for discontinuous CsCl density centrifugation as described in the text. Gradient fractions were assayed for PPV-specific hemagglutinating activity (HA) and density (A). The two peak regions of HA activity were individually pooled. The 1.39-g/ml density peak was subjected to discontinuous CsCl centrifugation a second time in a gradient identical to that used the first time (see the text) and assayed similarly (B). The 1.30-g/ml density peak was centrifuged in a second gradient of slightly lower density than the first. The pooled fractions from the first CsCl gradient were adjusted to a final CsCl density of 1.25 g/ml and layered onto an equal volume of CsCl solution having a density of 1.30 g/ml. Centrifugation and subsequent gradient fraction assay (C) were performed as described in the text. by various peptide mapping procedures (12, 19, 22, 34). In these systems, it appears that the peptides of the C protein are contained within the peptides of the B protein, which are in turn a subset of those of the A polypeptide. To determine whether a similar situation existed with the PPV structural proteins, we performed onedimensional partial proteolysis mapping studies on the three PPV polypeptides. E -p (0) z 0a

6 VOL. 45, 1983 PPV: PURIFICATION AND ANTIGENIC PROPERTIES 847 A. i,30g /ml 0 ol C. i I E ' B 92-, 68- -_ 45- _ -A _- C FIG. 3. Analysis of CsCl density gradient fractions containing PPV-specific hemagglutination activity. The and 1.30-g/ml peaks of HA activity (Fig. 2B and C, respectively) were individually pooled and extensively dialyzed against TE buffer. Portions of the dialyzed materials were analyzed by negative staining in the electron microscope (see the text). (A) 1.30-g/ml density material. (B) 1.39-g/ml density material. Bar, 40 nm. Another portion of the dialyzed materials was subjected to electrophoresis in an SDS-l0o polyacrylamide gel. The gel was then stained with Coomassie brilliant blue and destained (C). Molecular weight standards were included in an adjacent track in the gel (numbers represent kilodaltons). The proteins of highly purified PPV (1.39-g/ml particles) were radiolabeled in vitro with 1251 and separated by SDS-polyacrylamide gel electrophoresis. To ensure the purity of each polypeptide, the three proteins were individually subjected to a second cycle of polyacrylamide gel electrophoresis before any analyses were performed. A portion of each of the twice-gelpurified proteins was electrophoresed on a third polyacrylamide gel (Fig. 4a). Using the partial proteolytic mapping procedure originally described by Cleveland et al. (4), we compared the partial digests of the 1251_ labeled PPV A, B, and C proteins by using three different proteases: S. aureus V8 protease, elastase, and chymotrypsin (Fig. 4). The V8 protease patterns were nearly identical for the three polypeptides. The elastase and chymotrypsin patterns of each of the three proteins were also very similar, with many peptides from the three proteins migrating identically. It appeared, however, that several of the peptide bands derived from protein A migrated more slowly than the similar peptides in the B protein digest. Similarly, certain peptides in the B protein pattern migrated more slowly than the corresponding C protein peptides (Fig. 4). This suggests that these fragments are terminal portions of the respective proteins, which are otherwise very similar in primary structure. Use of individual PPV structural proteins to generate PPV-specific antisera. The data reported above suggest close structural similarities among the three virion polypeptides A, B, and C of PPV. To further compare these polypeptide, we felt it would be useful to prepare antisera to each individual SDS-denatured protein. To this end, highly purified PPV preparations were disrupted and subjected to preparative SDS-polyacrylamide gel electrophoresis to separate the three viral proteins. After localization of the protein bands, each was excised and then was subjected to a second cycle of gel electrophoresis as was done for the above-described 125I1 labeled proteins. The protein bands of the second polyacrylamide gel were localized and extracted from the gel pieces as described above. Figure 5 shows a Coomassie blue-stained polyacrylamide gel of aliquots of each of the gelpurified proteins and a sample of the starting virus preparation. After these procedures, we routinely were unable to detect contamination of

7 848 MOLITOR, JOO, AND COLLETT J. VIROL..; A (a) r. C: 0. * i.- II (b). 0 1** *i 43 F~rA. (c) S. *9. O.. * I; 9..0 _ '¼-;;..; F (d) sg -U FIG. 4. Partial proteolysis mapping of 125I-labeled PPV structural proteins. Purified virions (1.39 g/ml) were disrupted and radiolabeled with 1251 as described in the text. The three individual polypeptides, A, B, and C, were gel purified twice (see the text), and a portion of each was re-electrophoresed in a third SDS-10% polyacrylamide gel to assess their purity (panel a). Portions of each polypeptide were then subjected to partial proteolytic hydrolysis during electrophoresis in SDS-15% polyacrylamide gels as described previously (4), using (b) V8 protease, 0.1,ug per track; (c) elastase, 1,g per track; and (d) chymotrypsin, 5,ug per track. After electrophoresis, gels were dried and exposed to X-ray film. The numbers in the margins represent approximate molecular weights (in kilodaltons). one protein with the others, even when 1251 labeled proteins were similarly purified (Fig. 4). However, we do feel that contamination below the sensitivity of our tests could have existed. Each of these gel-purified proteins was used to immunize rabbits. In addition, intact PPV was used as an immunogen. At 3 and 7 weeks postimmunization, the rabbits receiving the gelpurified proteins were boosted with additional protein. At various times after the primary immunization, small samples of sera were obtained from each rabbit and assayed for PPV-specific antibodies by an HAI assay. The immune responses of each rabbit are shown in Fig. 6. All of the immunogens elicited a PPV-specific response; however, it appeared that intact virus was able to generate the strongest and most rapid response. It must be noted here, however, that only one rabbit was used for each immunization. We feel that this precludes any conclusions concerning the antigenic strength of the various immunogen preparations. A summary of the various antisera used in this study is provided in Table 1. Serological tests comparing various PPV-specific antisera. A variety of standard serological tests was performed, using conventional antisera to PPV and antisera generated by the SDSdenatured, gel-purified viral proteins. The results of these tests are summarized in Table 2. Although the antisera generated against the gelpurified proteins exhibited lower HAI titers than antisera produced from intact virus (Fig. 6; Table 2), these antisera did react with viral antigens in infected cells in an IFA assay and did contain precipitating antibodies as determined in an AGID assay. Ability of antisera to recognize denatured and native PPV antigens. We went on to compare the reactivities of the various PPV antisera to both denatured viral proteins and native, intact virus particles. Because antisera were generated

8 VOL. 45, 1983 C B A ST L..L..-..II. i IN~ 45- FIG. 5. Purity of PPV polypeptides A, B, and C used for rabbit immunizations. Purified virions (1.39 g/ ml) were disrupted by boiling in SDS-sample buffer and electrophoresed in preparative SDS-101% polyacrylamide gels. After localization (see the text), the three polypeptides A, B, and C were excised and then were individually subjected to a second cycle of electrophoresis. A portion of the three protein preparations obtained after gel elution and dialysis (see the text) was then electrophoresed in another gel together with a sample of the starting virus preparation (ST). The gel was stained with Coomassie brilliant blue and destained POST-IMMUNIZATION TIME (WEEKS) FIG. 6. Immune response of rabbits injected with intact virions and isolated PPV structural proteins. Individual rabbits were immunized with 50,ug of intact, CsCl-purified virus (1.39-g/ml particles), gelpurified polypeptide /A, gel-purified polypeptide B, and gel-purified polypeptide C as described in the text. Rabbits receiving gel-purified polypeptides were boosted at 3 and 7 weeks after the initial injection, and the rabbit receiving intact virus was boosted at 3 weeks only. At various times after the primary immunization, blood samples were collected, and the sera were assayed for PPV-specific antibodies by hemagglutination inhibition (see the text). PPV: PURIFICATION AND ANTIGENIC PROPERTIES 849 _ TABLE 2. Serological tests comparing the immune response of rabbits and swine immunized with either intact PPV or gel-purified structural proteins Serological test Antiserum resulta HAI IFA AGID Normal pig <4 - - Adult pig a PPV 8, Fetal pig a PPV 8, Normal rabbit serum <4 Rabbit a intact PPV 8, Rabbit a A polypeptide 1, Rabbit a B polypeptide Rabbit a C polypeptide 2, a The serological assays, HAI (hemagglutination inhibition), IFA (indirect fluorescent-antibody), and AGID (agar gel immunodiffusion), are described in the text. -, No detectable reactivity; +, clearly positive reactivity. As the IFA and AGID are difficult to evaluate quantitatively, no attempt is made here to distinguish antisera reactivities. against the SDS-gel-purified proteins, we might expect these sera to contain antibodies directed more toward linear determinants on the proteins, whereas antisera produced by immunization with intact virions might contain antibodies to higher-order structural determinants. PPV a- (f) 0- Go u z cr : c I2 4 5 FIG. 7. Reactivity of various antisera to denatured PPV polypeptides. Punified PPV was disrupted by boiling in SDS-sample buffer, and the viral polypeptides were resolved on an SDS-10% polyacrylamide gel. The proteins were then electrophoretically transferred to nitrocellulose paper (2), strips were cut, and individual strips were incubated with various antisera (see Table 1), followed by reaction with "N-I-abeled protein A (see the text). The washed strips were then exposed to X-ray film. The exposure time for all strips shown was the same (10 min). Upon longer exposure (5 to 6 h), a qualitatively similar reactivity of R a PPV with the PPV proteins was revealed; however, no reactivity was observed with NRS.

9 850 MOLITOR, JOO, AND COLLETT particles were disrupted by boiling in SDS and 2- mercaptoethanol, resolved by electrophoresis in an SDS-polyacrylamide gel, and transferred to nitrocellulose paper. Identical strips of the protein-containing nitrocellulose paper were then incubated with the various antisera, followed by incubation with 125I-labeled protein A to localized bound immunoglobulin. The results of this "Western blot" analysis are shown in Fig. 7. Normal rabbit serum showed no reactivity to the PPV proteins (Fig. 7, track 1). The individual antisera generated against each of the viral proteins showed very strong reactivity with all three PPV structural proteins (Fig. 7, tracks 3 to 5). Antisera obtained by immunization with intact virus reacted very poorly with the denatured antigens (Fig. 7, track 2). However, longer autoradiographic exposures (10 times) did reveal some reactivity to the three viral proteins. Similarly, porcine antisera obtained from naturally infected adult pigs or infected fetuses reacted poorly with the denatured PPV polypeptides (data not shown). Thus, as expected, antisera raised against the SDS-gel-purified proteins reacted very well with the denatured polypeptides. It is of interest here that all antisera reacted to all three viral structural proteins. Even though antiserum was generated by immunization with each individual viral protein, each antiserum, in addition to recognizing the immunizing antigen, recognized equally well the other two viral proteins. This result is consistent with the close structural similarities among the three proteins (Fig. 4). However, an alternate interpretation of these results is that our immunogen preparations of the gel-purified proteins were sufficiently contaminated with the other polypeptides that an immune response was also mounted against these contaminant proteins. To determine whether the various antisera were able to recognize native, intact virus particles, we tested their ability to cause the immunospecific aggregation (lattice formation) of PPV virions in an immunoelectron microscopy assay (7). Antisera were mixed with purified virus, and any aggregates formed were collected by centrifugation. The pelleted material was then analyzed after negative staining in the electron microscope. Immunospecific lattice formation occurred with antisera generated against native virus (Fig. 8, panel 2) and with the antisera generated against the individual gel-purified, denatured proteins (Fig. 8, panels 3 to 5), but not with normal, preimmune serum (Fig. 8, panel 1). Ability of antisera to neutralize virus infectivity. The results described above indicate that antisera produced by immunization with SDSgel-purified PPV proteins are able to recognize native, intact virus particles as well as linear determinants on the denatured viral polypep- J. VIROL. tides. We finally asked whether these antisera contained antibodies capable of neutralizing virus infectivity. Using a direct immunofluorescence assay, we found that antiserum generated by each of the gel-purified proteins did indeed contain neutralizing antibodies (Fig. 9; Table 3). It appears, however, that the titer of the neutralizing antibodies in each of the antiersa produced against the individual proteins was lower than that found in antisera generated against the intact virus. The neutralizing titers of the sera paralleled the HAI titers described above (Table 2). DISCUSSION Swine fetuses, either naturally exposed to or experimentally inoculated with PPV, actively support virus replication, which ultimately results in fetal death. As PPV is often difficult to grow to high titers in cells cultured in vitro, we initially concentrated on characterizing the molecular features of PPV as propagated in infected animals. Recently, a brief report on certain aspects of PPV replication in cultured fetal porcine kidney cells has appeared (29). The availability of both an animal model and an in vitro cell culture system for virus propagation provides an excellent opportunity for the detailed and controlled study of the molecular, immunological, and pathogenic characterization of PPV. Propagation of PPV in swine fetuses provides an abundant source of virus material. Employing procedures used for the purification of previously studied parvoviruses (33), we have been able to obtain highly enriched virion preparations. These preparations contained two predominant forms of the 20-nm virus particle: "full" (DNAcontaining) particles with a density in CsCl of 1.39 g-ml, and "empty" (DNA-deficient) particles with the lighter density of 1.30 g/ml. Three major polypeptides, designated A, B, and C, were consistently found in both of these virus species, having molecular weights of 83,000, 64,000, 60,000, respectively. Other parvoviruses have been shown to consist of three structural proteins with molecular weights in the following ranges: A, 93,000 to 73,000; B, 80,000 to 64,000; C, 67,000 to 56,000 (32). We therefore conclude that these proteins are the structural proteins of PPV. Polypeptide A routinely represented approximately 10% of the total viral protein in both the full and the empty viral particles. Polypeptide C appeared most abundant in the "full" particles, whereas the B protein was present in the greatest relative amounts in the "empty" particles (Fig. 3). All of these results are consistent with previous observations in other studied parvovirus systems (35). Earlier reports on work with rodent parvovi-

10 VOL. 45, 1983 PPV: PURIFICATION AND ANTIGENIC PROPERTIES 851 Downloaded from FIG. 8. Immunoelectron microscopy of purified PPV reacted with various antisera. Various antisera (see Table 1) were incubated with purified, intact virus particles and tested for their ability to form immune specific virus-antibody lattices as described in the text. ruses (34), the defective adeno-associated viruses (12, 19), and, more recently, with a densonucleosis virus (22) have shown that the A, B, and C viral structural proteins in their respective systems have extensive sequence homology with one another. It now appears to be accepted that the lower-molecular-weight polypeptides are subsets of the larger proteins; however, the means of their derivation and the mechanism of putative RNA or protein processing remain obscure. In our limited structural analyses of the PPV proteins, we found that the three viral proteins were also very closely related to each other in primary sequence. A variety of antisera were generated to investigate certain antigenic properties of PPV and its constituent proteins. Our greatest interest centered on the antisera that were raised against the SDS-denatured, gel-purified viral structural proteins. Antibodies elicited by the individual gel-purified polypeptides were qualitatively similar to antibodies in sera from naturally infected adult pigs or rabbits experimentally immunized with intact virus particles in several standard serological assays, including HAI, an IFA test, and an AGID assay (Table 2). However, the antisera generated against the denatured, gelpurified proteins reacted more avidly with the denatured antigens than did the conventional antisera (Fig. 7). Still, these antisera were all able to recognize and immunospecifically aggregate native intact virion particles (Fig. 8). Finally, all antisera directed against PPV, whether produced by natural infection, immunization on November 15, 2018 by guest

11 852 MOLITOR, JOO, AND COLLETVJ. VIROL. Downloaded from FIG. 9. Virus infectivity neutralization by various antisera detected by direct immunofluorescence. Various sera (diluted 1:10 in PBS) were mixed and incubated with PPV and then were placed on cultures of ST cells, as described in the text. After 4 days, PPV cellular antigens were detected with a fluorescent-antibody conjugate and viewed under a fluorescent microscope (see the text). The sera used were (1) NRS; (2) R a PPV; (3) R a A; (4) R a B; (5) R a C (see Table 1 for abbreviations). with intact virus particles, or immunization with the individual, SDS-gel-purified viral structural proteins, were able to neutralize virus infectivity (Fig. 9; Table 3). Antisera prepared against individual parvovirus structural proteins in other systems have failed to elicit a virus-neutralizing response (6, 9). Several explanations may account for this apparent discrepancy. We feel that the method of antigen preparation may be an important factor. We find it interesting that each of the antisera generated against the individually gel-purified viral proteins reacted with all three structural proteins and that each viral polypeptide was capable of eliciting virus-neutralizing antibody. These results are consistent with the sequence homology among the three proteins and suggest that the virus-neutralizing determinant(s) is present on all three viral structural proteins. However, we feel that due to the possible minor contamination of each immunogen preparation with the other structural proteins, and to the fact that the neutralizing antibody response could have been a result of such contamination, any conclusions concerning the nature of PPV-neutralizing antigenic determinants is unwarranted at this time. The generation of monoclonal antibodies to these proteins should provide more conclusive information concerning the number and distribution of virus-neutralizing antigenic on November 15, 2018 by guest

12 VOL. 45, 1983 TABLE 3. Viral infectivity neutralization of various antisera Culture fluid Fluorescent antigen Antiseruma (HA)b cells (%)C 1:2 1:10 1:2 1:10 No serum No virus 256 <4 256 <4 >80 0 >80 0 Normal pig serum >80 >80 Fetal pig a PPV <4 <4 0 0 Normal rabbit serum >80 >80 Rabbit a intact PPV <4 <4 0 0 Rabbit a A <4 < Rabbit a B < Rabbit a C < a For standardization, rabbit a A, a B, and a C antisera were normalized to the same HAI titer and then were diluted either 1:2 or 1:10 in PBS. b Cell culture fluids were tested at 3 days postinfection for the presence of extracellular (culture fluid) virus by hemagglutination of guinea pig erythrocytes (see the text). c Acetone-fixed cells were stained at 3 days postinfection with PPV-fluorescent antibody conjugate for the presence of intracellular virus as described in the text. The percentage offluorescent cells was estimated from at least five independent fields of view. determinants on the PPV structural polypeptides. 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