Veterinary Microbiology, 32 (1992) 281-292 Elsevier Science Publishers B.V., Amsterdam

281

Recombinant polypeptide from the gp48 region of the bovine viral diarrhea virus (BVDV) detects serum antibodies in vaccinated and infected cattle J i m m y K w a n g a, E. Travis Littledike a, R u b e n O. D o n i s b a n d E d w a r d J. D u b o v i c aUSDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE, USA bDepartment of Veterinary Science, University of Nebraska, Lincoln, NE, USA CDiagnostic Laboratory, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY, USA (Accepted 28 January 1992)

ABSTRACT

Kwang, J., Littledike, E.T., Donis, R.O. and Dubovi, E.J., 1992. Recombinant polypeptide from the gp48 region of the bovine viral diarrhea virus (BVDV) detects serum antibodies in vaccinated and infected cattle. Vet. Microbiol., 32:281-292. To characterize the immune response of cattle to bovine viral diarrhea virus (BVDV) glycoprotein gp48, we have produced a large amount of recombinant glutathione-s-transferase-gp48 (GST-gp48) fusion protein in Escherichia coli. Antibodies to gp48 were present in cattle vaccinated with killed or modified-live virus vaccination, or following natural infection. These results were in agreement with results of serum neutralization (SN) test which detected gp53 of BVDV.

INTRODUCTION B o v i n e v i r a l d i a r r h e a v i r u s ( B V D V ) is a n e n v e l o p e d s i n g l e - s t r a n d e d R N A v i r u s t h a t is the p r o t o t y p e for t h e P e s t i v i r u s g e n u s o f the f a m i l y F l a v i v i r i d a e ( W e s t a w a y et al., 1985; Collett et al., 1988b; C o l l e t t et al., 1989). T h i s g e n u s also i n c l u d e s the i m p o r t a n t serologically r e l a t e d h o g c h o l e r a v i r u s ( H C V ) a n d b o r d e r disease v i r u s ( B D V ) in s h e e p ( D a r b y s h i r e , 1960; D i n t e r , 1963; O s b u r n et al., 1973 ). B o v i n e viral d i a r r h e a h a s b e e n r e c o g n i z e d in m a n y p a r t s o f t h e w o r l d a n d has a m a r k e d e c o n o m i c i m p a c t o n the cattle i n d u s t r y ( B r o w n l i e , 1985). Correspondence to: J. Kwang, USDA, ARS, U.S. Meat Animal Research Center, Clay Center, NE 68933, USA. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.

282

The cDNA cloning of the BVDV mRNA led to important advances in understanding the genomic organization of BVDV, and a protein encoding map of the BVDV genome was established (Renard et al., 1987; Collett et al., 1988a). According to this map, the putative glycoprotein precursor gpl 16, encoded near the 5' end of the genome, gives rise to gp62 and gp53 through proteolytic processes; and gp62, is subsequently cleaved to gp48 and gp25. Thus, the order of the genes encoding this region of BVDV glycoproteins is gp48-gp25-gp 53 (Stark et al., 1990). Based on the radioimmunoprecipitation (RIP) results of cattle to individual BVDV proteins, a strong immune response to glycoproteins gp53 and gp48 was noticed (Donis and Dubovi, 1987a,b). This suggests that both glycoproteins are among the immunodominant viral proteins in infected cells. Although much work has been reported on interaction of gp53 protein and host immune system (Donis and Dubovi, 1987a,b; Bolin et al., 1988; Magar et al., 1988; Bolin and Ridpath, 1990), little is known about the immune response to gp48. The gp48 gene product has been expressed as a fusion protein with fl-galactosidase in bacteria and this fusion protein immunoprecipitated gp116, gp62, and gp48 antibodies (Collett et al., 1988a). To allow study of the role of the BVDV-gp48 during infection and the subsequent immune response in cattle, we generated constructs that expressed the gp48 as a fusion protein with glutathione-s-transferase (GST). Recombinant GST-gp48 fusion protein was produced, partially purified and a specific Western blot assay was developed. This assay was used to test for antibodies to gp48 in vaccinated and naturally infected cattle. We examined 175 serum samples and found a complete correlation between results obtained by serum neutralization ( SN ) test and by GST-gp48 western blotting. These results indicated the application of recombinant gp48 polypeptide may be useful to detect BVDV antibodies in bovine sera. MATERIALS AND METHODS

Source o f bovine sera A total of 175 bovine sera were included in this study: 80 samples from cattle at the U.S. Meat Animal Research Center (MARC) and 95 samples from an isolated commercial ranch in north-central Nebraska. The MARC herd has been on both killed and modified-live virus vaccination programs for the last 10 years; whereas, the cattle at the commercial farm had never been vaccinated with BVD vaccines. Therefore, antibody-positive individuals might represent the outcome of either/or various combinations of, natural infection, killed-virus vaccination, modified-live virus vaccination or passive immunity from the mother. The sera were first examined for BVDV neutralizing antibodies (TGAC strain) to determine the proportion of virus seropositive individuals. The SN testing was carried out as previously described

283 (Bolin et al., 1988 ). Five serum samples, testing negative for BVDV by the SN test and RIP (both with TGAC strain), were used as the negative reference. Two immunized cattle sera (anti-NADL and anti-Nebraska) were used as positive reference sera.

Bacterial strain and plasmids Escherichia coli JM105 was the host strain for all the recombinant plasmids. Procedures for purifying plasmid DNA, DNA manipulations, bacterial transformations, and DNA sequencing reaction have been previously described (Maniatis et al., 1982 ). Plasmid pBV4-gp62 containing BVDV-NADL genome from nucleotides 1113 to 2480, which probably covers the entire gp62 gene, was the basis for further constructs (Collett et al., 1988). Plasmid pGex3x (Pharmacia, Piscataway, N J) was used as starting material for constructing the gp48 expression vector. The pGex3x vector contains the GST gene under the control of the Tac promoter. We chose pGex3x vector to express gp48 as a fusion protein with GST since the pGST expression system may offer 1 ) ease of purification of fusion protein through using glutathione affinity column and 2 ) factor Xa cleavage site for proteolytic removal of GST from GST-gp48 fusion proteins following their purification (Smith and Johnson, 1988).

Monoclonal antibodies to gp48 Production and characterization of the mouse monoclonal antibodies to BVDV-gp48 have been recently reported (Corapi et al., 1988, 1990; Donis et al., 1988).

SDS-polyacrylamide gel electrophoresis and western blotting These procedures were performed by the standard protocols (Laemmli, 1970; Burnett, 1981 ).

Preparation of bacterial lysates for gel analysis JM 105 bacteria, containing the vector with the gp48 DNA insert (pGex3xgp48 ), were grown to an optical density of 0.6 at 600 nm, in 2 ml L broth with 50/Lg/ml ampicillin. The cultures were induced by 1 mM isopropyl-fl-D-thiogalactopyranoside (IPTG) for 4 h. Uninduced cultures were grown under identical conditions without the addition of IPTG. Bacterial cells were isolated by centrifugation at 4000 g for 10 min. The resulting cell pellets were suspended in 180/zl SDS-loading buffer ( 10% glycerol, 50 mM dithiothreitol, 3% SDS, 0.0625 M Tris hydrochloride [pH 6.8 ], 0.02% bromophenol blue) and 10/tl of each preparation were analyzed on PAGE gel.

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Purification of polypeptides One liter of JM105 cells bearing pGex3x-gp48 were grown to stationary phase at 37 °C and induced as described above. The cells were harvested by centrifugation and washed once with 10 mM Tris-HC1 buffer (pH 7.5). The cell pellets were then resuspended in 4 vols of 50 mM Tris-HC1 (pH 8.0) 0.5 mM EDTA - 0.3 M NaC1 - 1 m g / m l of lysozyme and incubated for 15 min on ice. At the end of incubation, the cells were lysed with 0.5% nonidet p40. The lysates were centrifuged at 12 000 g for 5 min and the supernatant designated S1. Extraction of GST-gp48 from the pelleted material resulting from the cell lysis was done in three steps. Step 1, the pellet resulting from the bacterial cell lysis was suspended in 8 ml 1 M urea buffer ( 1 M urea, 25 mM Tris [pH 8.0], 10 m M EDTA), centrifuged, and the supernatant was saved (designated the $2 fraction). Step 2, the pellet was further extracted with 8 ml 6 M urea buffer, centrifuged, and the supernatant designated fraction $3. Step 3, the remaining pellet was extracted with 8 ml SDS-loading buffer, centrifuged, and the supernatant fraction designated $4. Although a small amount of GST-gp48 was left in $3, most of it was recovered in $4 supernatant (Fig. 2B, Lanes 4 and 5 ). RESULTS

Construction of pGex3x-gp48 expression vector A 359 bp Hae III fragment of the pBV4-gp62 plasmid, corresponding to the nucleotides 1295 to 1653 of the BVDV-NADL genome, was cloned into the 3' end of the GST gene (Fig. 1A). A plasmid having the gp48 sequence in phase with the ATG start codon on the GST sequence was designed and designated pGex3x-gp48. The correct construction joining the GST and gp48 sequence in the same reading frame was verified by DNA sequence analysis across the GST-gp48 junction (Fig. 1B). The pGex3x-gp48 expression vector theoretically directed a 39 kDa protein. This molecular mass was calculated from the sum of the GST (26 kDa) and gp48 (13 kDa) sequences inserted into the expression vector. The pGex3x-gp48 expression vector was introduced into JM105 bacteria. When grown under the condition as described in the Material and Methods, this construct was found to express high levels of a 39 kDa fusion protein. Figure 2A shows total cell lysates of the E. coli strain producing GST-gp48 protein. The 39 kDa protein was the major protein in total cell lysates and accounted for approximately 10% of the total protein as estimated by SDSPAGE. This protein was not observed in either uninduced cells containing pGex3x-gp48 plasmid, or induced cells containing the pGex3x vector without an insert. The size of the 39 kDa protein coincides with that predicted for the expected fusion protein. Besides the 39 kDa major protein, a smaller protein (approximately 30

285

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Fig. 2. Expression, purification, detection, and absence of cross-reactivity of the GST-gp48 fusion protein. Panels A and B: Coomassie blue stained gel, and Panel C and D: Western blot. (A) Total cell lysate: 1, Uninduced control lysate; 2, 3 h induction lysate; 3, 3-1/2 h induction lysate; 4, 4 h induction lysate; 5, Protein molecular weight standards from BRL - high range. Ten/~1 of each preparation was loaded on each lane. The GST-gp48 band is indicated by an arrow and the breakdown product is indicated by an asterisk. (B) Purification of GST-gp48: 1, Total induced lysate; 2, S1 fraction (lyric supernatant); 3, $2 fraction ( 1 M urea supernatant); 4, $3 fraction (6 M urea supernatant); 5, $4 fraction (sample buffer supernatant). Ten/zl of each preparation was loaded on each lane. (C) Detection of GST-gp48: 1, Monoclonal antibody (15C5) to gp48; 2, NADL strain immunized cattle serum; 3, Nebraska strain immunized cattle serum; 4, BVDV infected cattle serum; 5, Pooled BVDV negative serum. Background band, presumably due to anti-E, coli antibodies present in most bovine serum. Western blot assays were performed, as described in the Materials and Methods. (D) Confirmation of the absence of cross-reactivity with GST moiety: ( 1 ), (2), and (3) western blots of GST; and (4) western blot of GST-gp48. These western blot strips were reacted with: 1, BVDV positive serum; 2, BVDV negative serum; 3 & 4, Guinea pig anti-GST serum.

287 kDa) was also present in SDS-PAGE gel (Fig. 2A ). To test whether the smaller protein was the breakdown product of the 39 kDa protein, we removed cultures at 3 h, 3-1/2 h, and 4 h intervals after induction. At 3 h of induction, the intact fusion protein was observed without the production of breakdown products (Fig. 2A, Lane 2). Accumulation of the breakdown product at 30 kDa with the increasing length of induction time, suggests that the 30 kDa smaller protein is degradation of the primary translation product by protease within the cell (Fig. 2A, Lanes 3 and 4).

Confirmation of GST-gp48 protein The identity ofgp48 product in E. coli as a fusion protein was examined in 4 ways by western blot (Fig. 2C and 2D): • This fusion protein was recognized by the specific monoclonal antibody to gp48 (Fig. 2C). • Immunized cattle sera against NADL and Nebraska strains reacted with the expressed protein. BVDV infected cattle sera also recognized the gp48 moiety of the fusion protein (Fig. 2C). • Immunoblots with pooled BVDV negative sera did not react with the expressed protein (Fig. 2C). • To confirm the absence of cross-reactivity of BVDV antisera with GST moiety (Fig. 2D). BVDV positive or negative sera did not react with bacterial cell extracts containing the GST only. However, antibodies raised in a guinea pig (unpublished data) against purified GST recognized 26 kDa GST band and 39 kDa GST-gp48 band on the GST and GST-gp48 western blot, respectively. These results, together with sequence data, confirmed that the 39 kDa protein was the product of the expressed GST-gp48 in E. coli.

Reactivity of antibodies in bovine sera with GST-gp48fusion protein Serum neutralization test results of 175 bovine sera were summarized in Table 1. When these results were tabulated, 89.1% of the serum samples had antibodies to BVDV and 10.9% had a negative SN titer. The 175 bovine sera were subjected to the GST-gp48 western blot assay. Sera from 156 animals with SN titer >~1:4 produced a prominent reactive band at 39 kDa in GSTgp48 western blots; whereas, none of the 19 sera with a negative SN test ( ~/1:256

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