key: cord-007476-wu9tuvy9 authors: Katz, Jonathan B.; Shafer, Amy L.; Eernisse, Kenneth A.; Landgraf, John G.; Nelson, Eric A. title: Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) are encoded by the carboxyterminal portion of viral open reading frame 3 date: 2000-03-10 journal: Vet Microbiol DOI: 10.1016/0378-1135(94)00113-b sha: doc_id: 7476 cord_uid: wu9tuvy9 Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were revealed by serologic analysis of a recombinant protein derived from PRRSV open reading frame 3 (ORF 3). The hydrophilic carboxyterminal 199 amino acids encoded by the ORF 3 of a European (Lelystad) isolate of PRRSV were expressed as a recombinant fusion protein (BP03-P) in a baculovirus gene expression system. Sera from gnotobiotic swine exposed to prototypic reference European and American isolates of PRRSV and sera from conventionally reared European and American swine convalescing from naturally acquired PRRSV infections were used to characterize the BP03-P protein. Sera from gnotobiotic and conventionally reared swine exposed to European isolates of PRRSV were significantly more reactive (P < 0.01) with BP03-P than were the corresponding American PRRSV antisera using the indirect immunoperoxidase monolayer assay (IPMA). Prototypic European, but not American, PRRSV antisera also recognized BP03-P using western immunoblotting and radioimmunoprecipitation assay (RIPA) procedures. However, gnotobiotically derived antiserum to an atypical American-origin PRRSV was reactive with BP03-P by both IPMA and western immunoblot. Despite a predicted potential for N-linked glycosylation, studies with tunicamycin and peptide-N-glycosidase F (PNGase F) indicated that BP03-P was not N-glycosylated in either insect cell cultures or Trichoplusia ni larvae infected with the recombinant baculovirus. Sera from rabbits inoculated with BP03-P failed to neutralize both the European (Lelystad) and American (ATCC VR-2332) reference isolates of PRRSV and did not react by IPMA with PRRSV-infected cell cultures. Taken together, the data suggest that the carboxyterminal portion of PRRSV ORF 3 encodes a non-neutralizing viral peptide that is partially responsible for the serologic differences noted between European and most American isolates of PRRSV. F ( PNGase F) indicated that BPO3-P was not N-glycosylated in either insect cell cultures or Trichoplusia ni larvae infected with the recombinant baculovirus. Sera from rabbits inoculated with BPO3-P failed to neutralize both the European (Lelystad) and American ( ATCC VR-2332) reference isolates of PRRSV and did not react by IPMA with PRRSV-infected cell cultures. Taken together, the data suggest that the carbox- In the few years since it was initially described (Keffaber, 1989; Lindhaus and Lindhaus, 199 1; Paton et al., 1991) , the disease complex now known as the porcine reproductive and respiratory syndrome (PRRS) has become an economically significant swine health problem throughout Europe and North America (Goyal, 1993) . The viral etiology of PRRS was confirmed both in The Netherlands (Meulenberg et al., 1993; Terpstra et al., 1991) and in the United States with the isolation of two previously unknown PRRS viruses (PRRSV), a prototypic European PRRSV (Lelystad isolate) and a prototypic American PRRSV (VR-2332 isolate). These 50 to 60 nm enveloped RNA viruses Wensvoort et al., 1992) share common antigenic determinants but may be differentiated using both polyclonal antisera and monoclonal antibodies (Nelson et al., 1993) . Serologic analysis of European and American swine naturally exposed to PRRSV has confirmed that geographically based antigenic differences do exist between most North American and most European isolates of PRRSV; these differences are consistent with those first noted between the Lelystad and the VR-2332 isolates Bautista et al., 1993) . There is some evidence, however, that antigenically intermediate PRRSV or possibly both prototypic antigenic types may exist within the North American swine population (Bautista et al., 1993) . PRRSV has been tentatively classified as an arterivirus, along with equine arteritis virus (EAV) and two other proposed members of that genus: lactate dehydrogenase elevating virus (LDV) and simian hemorrhagic fever virus (SHFV) (Plagemann and Moennig, 1992) . PRRSV contains a 12 to 14 Kb single strand positive polarity polyadenylated genome (Plagemann and Moennig, 1992) and utilizes a nested 3' coterminal mR.NA gene expression strategy (Meulenberg et al., 1993; Conzelmann et al., 1993) . The PRRSV genome contains 8 open reading frames (ORFs), including the large 5' terminal ORFs la and lb encoding the putative viral polymerase and the 3' terminal ORF 7 encoding the nucleocapsid protein (Meulenberg et al., 1993; Conzelmann et al., 1993) . PRRSV virions are believed to contain two other structural, presumably envelope, proteins (Nelson et al., 1993) . It has not been determined which of the ORFs 2 through 6 encode these proteins or the identities and functions of the other proteins encoded by these ORFs. Recombinant DNA technology permits the expression of individual gene segments in order to better understand the antigenicity of their protein products and their roles in the host-virus relationship. We report here the expression and serologic analysis of a markedly hydrophilic portion of PRRSV ORF 3 (Lelystad isolate). This segment was of additional interest because comparison of LDV, EAV, and PRRSV ORFs 2 through 6 revealed that the ORF 3 protein products of these viruses would be the most divergent in size but potentially the most universally highly N-glycosylated of all the proteins encoded by those ORFs (den Boon et al., 199 1; Conzelmann et al., 1993; Godeny et al., 1993) . These features suggested to us that PRRSV ORF 3 might encode a glycoprotein mediating virus-host cell interactions; therefore, it might be a target of host defense mechanisms subjected to selective evolutionary pressure. We hypothesized that it might account in part for the geographically oriented antigenic differences between the majority of European and North American PRRS viruses, and that was the basis for this study. The Lelystad and VR-2332 isolates of PRRSV were provided by C. Terpstra, Central Diergeneeskundic Institut, The Netherlands, and by D. Chladek, Boehringer Ingelheim Inc., USA, respectively. A British reference PRRSV isolate, Humberside 2, was a gift from S. Edwards, Central Veterinary Laboratory, United Kingdom. PRRSV isolates 46448 and 27 199 were recovered in this laboratory from midwestem American swine tissues. Antisera to the 46448 isolate had been found uniformly cross-reactive with numerous North American PRRS virus isolates; isolate 27 199 was of interest because it was completely fastidious for replication in swine pulmonary alveolar macrophages. All viruses except 27 199 were propagated in cultures of MARC-145 cells, which are a PRRSV permissive cell line subcloned from MA-104 cells (Kim et al., 1993) . The identity of all viruses was confirmed using PRRSV-specific monoclonal antibodies in an indirect immunoperoxidase monolayer assay (IPMA) format. Gnotobiotic swine were used to prepare antisera to each PRRSV isolate. Each lo-dayold piglet was intranasally inoculated with lo5 to lo6 TCID,, of one virus isolate, and the resultant convalescent antiserum was collected between 48 to 56 days post-inoculation. Twenty field origin sera derived from clinical cases of PRRS in swine from northern Germany were generously provided by T. Blaha, Tierartzliche Hochschule, Hanover, Germany. Fifteen American field origin PRRSV seropositive swine sera were also obtained from D. Kinker, Iowa State University, Ames, IA, USA. Each serum originated from a separate swine herd experiencing PRRS, and these sera represented samples from herds located in 11 states in the eastern, southern, and midwestem United States. The IPMA ( Holm-Jensen, 198 1) was used to evaluate serial 4-fold serum dilutions for reactivity against both the Lelystad and the 46448 PRRSV isolates. These viruses had been inoculated 48 h previously onto separate MARC-145 cell monolayers. Immunodetection was accomplished using protein G-horseradish peroxidase (HRP) conjugate and aminoethylcarbazole substrate (Zymed, So. San Francisco, CA, USA). A PRRSV serum neutralization (SN) procedure was used to evaluate selected sera as recently described (Yoon et al., 1994) . Recombinant ORF 3 antigen, cesium chloride purified PRSSV (Nelson et al., 1993) , and ultrafiltered concentrates of cell cultures infected with the Lelystad isolate were used as antigens in western immunoblotting following denaturing polyacrylamide gel electrophoresis in 10% to 15% or 8% to 25% gradient gels (Laemmli, 1970; Burnett, 1981) . Immunodetection was again accomplished with protein G-HRP conjugate and an insoluble tetramethylbenzidine (TMB) substrate (Kirkegaard and Perry Inc., Gaithersburg, MD, USA). A radioimmunoprecipitation assay (RIPA) recently described for PRRSV (Nelson et al., 1993) was used with the same antigens to evaluate sera from the gnotobiotic swine inoculated with PRRSV isolates. A hydrophobicity/hydrophilicity plot (Kyte and Doolittle, 1982) of the putative ORP 3 protein revealed a markedly hydrophobic amino terminus followed by the remaining predominantly hydrophilic 75% ( 199 of 265 amino acids) of the protein (Fig. 1) . We postulated that this latter portion might be externalized and, therefore, available to interact with host cell receptors or antibodies and that the amino terminus perhaps represented a biosynthetically cleaved leader peptide or otherwise cryptic segment not directly interactive with the host. To focus our examination on the hydrophilic body of the protein, we excluded the hydrophobic segment through a subunit cloning strategy. Total RNA was extracted from a 25 cm* cell monolayer infected with the Lelystad isolate of PRRSV. A 20-mer oligonucleotide complementary to the viral RNA sequence (Meulenberg et al., 1993) 58 nucleotides downstream from the ORE 3 termination codon (positions 13,249 to 13,230 inclusive) was used to prime viral cDNA synthesis in a 20 ~1 reaction (Maniatis et al., 1982) . A 655 base pair (bp) cDNA segment was then amplified through 20 cycles of the polymerase chain reaction (PCR) (Saiki et al., 1988 ) using a 5' primer located between positions 12,559 to 12,578 and a 3' primer (13,213 to 13,194) to which a Pst I recognition sequence had been appended (Fig. 1) . Following Xho I and Pst I digestion and gel purification, the PCR product was directionally ligated into a baculovirus polyhedrin gene transfer plasmid, pAcSG-HIS-NT (Pharmingen, Inc., San Diego, CA, USA). The resulting plasmid contained the 199 carboxyterminal amino acid-encoding portion of ORF 3 fused in frame to a 5' vector sequence encoding 27 amino acids including a polyhistidine domain. The resulting fusion polypeptide had a calculated mass of 25,984 daltons (26 kDa) . Following DNA sequence verification, recombinant plasmid DNA ( 2 ug) was cotransfected with Bsu 361-digested AcNPV baculoviral DNA (0.5 ug) (Pharmingen, Inc.) into a Spodopteru frugiperda SF-9 cell culture using a liposomal transfection reagent (DOTAP, Boehringer Marmheim, Indianapolis, IN, USA). Clonally purified baculoviruses expressing PRRS virus-immunoreactive material were identified by IPMA. Separate PCRs of cell culture fluids from monolayers infected by these cloned viruses were used to confirm the presence of the ORF 3 gene sequence and the absence of the nonrecombinant baculovirus polyhedrin gene sequence. The latter PCR employed primers spanning the polyhedrin gene insertion site (Invitrogen, Inc., San Diego, CA, USA). One rDNA baculovirus, BP03, was used for recombinant protein production. Recombinant protein (BPO3-P) was produced by infecting both SF-9 cell cultures and 8-day-old Trichoplusia ni larvae according to standard procedures ( O'Reilly et al., 1992) . ] asparagine amidase (Boehringer Mannheim, Inc.) in a further effort to evaluate the N-glycosylation status of the protein. The polyhistidine sequence within BP03-P enabled efficient affinity purification of both cell culture and larval origin BPO3-P using immobilized nickel ion chromatography as previously described (Janknecht et al., 199 1) . Purified larval and cell culture origin BPO3-P was adjuvanted (Fatunmbi et al., 1992) with avridine (Pfizer, Inc., Groton, CT, USA) and used to immunize four rabbits each on four biweekly occasions. Sera from these animals were then evaluated by IPMA, SN, western blotting, and RIPA for anti-PRRSV reactivity. BF'O3-P expression was detected in the cytoplasm of infected SF-9 cells using gnotobiotically-derived Lelystad isolate antiserum in an IPMA format (Fig. 2) . BPO3-P expression was noted within 72 h post-infection (hpi) and persisted at least to 120 hpi. Comparison of BP03 infected and uninfected cell culture proteins by SDS-PAGE revealed the presence of a new protein in the infected cell cultures (Fig. 3A) . The size of this protein was consistent with the 26 kDa mass predicted from the amino acid sequence of the fusion protein encoded by BP03. The PRRSV-specific identity of this protein was confirmed by western immunoblotting (Fig. 3C ). BPO3-infected insect larvae contained a similarly sized immunoreactive protein within 96 hpi (Figs. 3B, 3C). Protracted incubation of affinity purified larval BP03-P with PNGase F did not result in a detectable reduction in apparent BPO3-P molecular mass (Fig. 3C ). There was also no detectable decrease in the molecular mass of BP03-P from tunicamycin treated cell cultures relative to untreated controls (Fig. 3C ). The IPMA, SN, RIPA, and western immunoblot procedures were used to evaluate rabbit antisera developed against both larval and cell culture origin BPO3-P. None of these sera exhibited respectively. Lane 2: Molecular mass markers (kDa) Panel C: Immunoblot of affinity purified larval BPO3-P protein with and without prior digestion with PNGase F (lanes 1 and 2, respectively). Immunoblot of SF-9 cell culture origin BPO3-P protein produced in the presence or absence of tunicamycin (lanes 3 and 4, respectively). Lane 5: Cell culture control infected with recombinant baculovirus containing vector-only transfer plasmid sequences. PRRSV SN activity at a 1:8 dilution. All sera were IPMA-reactive at 2 1:270 dilution using BP03-infected SF-9 cells but failed to react at a 1:20 dilution with 46448 isolate-infected MARC-145 cells. These sera also did not react at the 1:20 dilution with Lelystad isolateinfected cell monolayers fixed between 8 and 48 hpi. Two of the rabbit antipeptide sera were reproducibly reactive by western immunoblot with a diffuse (40 to 45 kDa) band of antigen found in homogenates of MARC-145 cells infected 16 h previously with the Lelystad isolate (Fig. 4) . Repeated RIPA and western blot attempts using these sera were unsuccessful in identifying a virus-specific protein in purified virion preparations. Antisera to gnotobiotic Lelystad, Humberside 2, and 27 199 virus isolates reacted specifically at 1: 100 dilutions with nitrocellulose blotted bpO3-p, while antisera to vr-2322 and 46448 virus isolates were totally nonreactive at the same or lower dilutions (Fig. 3B) . RIPA results were consistent with these findings. These sera were also evaluated by IPMA against Lelystad and 46448 infected Marc-145 cells and BPO3-infected SF-9 cells (Table 1) . Antisera to the Lelystad and Humberside 2 isolates were differentially reactive with cells 1 2345 6 Fig. 4 . Western immunoblot analysis of sera from two rabbits inoculated with BPO3-P. Lane 1: Molecular mass markers &Da). Lanes 2 and 3: Blots of rabbit anti-(BP03-P) sera, showing reactivity against 40 to 45 kDa proteins found in cells infected with PRRSV (Lelystad isolate) 16 hours previously. Lane 4: Blot of rabbit serum (same as used in Lane 3) against uninfected cell antigens. Lanes 5 and 6: Blots of hyperimmune anti-BPGf-P rabbit serum and gnotobiotic anti-PRRSV (Lelystad) swine serum, respectively, against the BPO3-P immunogen. Hyperimmune rabbit serum also detects small ( 18-20 kDa) breakdown products of BP03-P resulting from its catabolism in infected insect cells. 'German sera significantly more reactive (P < 0.0 1) with Lelystad and BP03 viral antigens than the corresponding American sera and significantly (P