Preliminary characterization of protein binding factor for porcine reproductive and respiratory syndrome virus on the surface of permissive and non-permissive cells.

In its natural host, porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to have a restricted tropism for cells of the monocyte/macrophage lineage. To date, cloned monkey kidney cell lines, such as MARC-145 and CL2621 cells which have been established from MA-104 cells, are the only non-porcine cells known to support PRRSV replication. In the present study, a binding assay was set up to follow by flow cytometry the attachment of PRRSV on the surface of porcine and non-porcine cells. PRRSV was found to be able to bind permissive cells like porcine alveolar macrophages and MARC-145. Further binding assays with porcine peripheral blood leukocytes showed that only monocytes can attach the virus. By their lack of binding factor, lymphocytes appeared to be refractory to PRRSV infection. Pre-incubation of MARC-145 cells with chymotrypsin and pronase E, but not neuraminidase, blocked their binding activity for PRRSV. The binding activity of the protease-treated cells was regenerated 8 hours after treatment, but cells remained unable to bind PRRSV if maintained in the presence of cycloheximide, thus confirming the proteinic nature of the specific binding factor(s). Experiments conduced with cells that have been previously characterized as non-permissive to PRRSV infection showed that many of them were able to bind the virus. Data obtained suggest that interaction of PRRSV with a specific binding factor on the surface of some cells is not sufficient to lead to a productive infection, and that a second putative receptor or other phenomena are probably required to pursue later events.

Competitive ELISA for detection of antibodies to porcine reproductive and respiratory syndrome virus using recombinant E. coli-expressed nucleocapsid protein as antigen.

The 15 kDa nucleocapsid (N) protein is the most abundant protein of the porcine reproductive and respiratory syndrome virus (PRRSV), and is highly antigenic, which therefore makes it a suitable candidate for the detection of virus-specific antibodies and diagnosis of the disease. In this study, complementary DNA corresponding to the entire N gene of the IAF-Klop strain of PRRSV was cloned into the pGEX-4T-1 vector, and the N protein was expressed in Escherichia coli fused to the glutathione S-transferase (GST) protein. The resulting GST-N recombinant fusion protein was purified by affinity chromatography and used as antigen for serological testing by indirect enzyme-linked immunosorbent assay (ELISA). Two anti-N specific monoclonal antibodies (MAbs) (IAF-K8 and IAF-2B4), obtained following fusion experiments with spleen cells of BAlb/c mice that were immunized with the purified virus, were used in a competitive assay to increase the specificity of the ELISA. Both MAbs were found to be directed against highly conserved conformational epitopes of North American isolates of PRRSV. Optimal concentration of GST-N protein was determined by checkerboard titration, using hyperimmune pig antiserum to the homologous PRRSV strain, and corresponded to a range of 0.1-0.5 microg protein per well. When tested on 95 sera from pigs that were experimentally infected with the IAF-Klop strain, the competitive ELISA (K8-ELISA) was capable of detecting anti-PRRSV antibodies in 86.7% (65/75) and 92.6% (63/68) of pig sera known to be seropositive by indirect immunofluorescence (antibody titers >16) and a currently used commercial ELISA (HerdCheck(R); Idexx), with specificity values of 100 and 96.2%, respectively. When tested on clinical samples (542 sera) from 28 positive and 28 negative pig herds, the K8-ELISA performed in a similar way to HerdCheck(R) and immunofluorescence (IF) tests as shown by kappa values of 0.762 and 0.803. The sensitivity and specificity of K8-ELISA were 100% on a herd basis, whereas sensitivity values of 80 and 82% with a specificity of 98.7% were determined on an individual basis in comparison with HerdCheck(R) and IF tests.

In vitro and in vivo inhibition of complement activity by a single-chain Fv fragment recognizing human C5.

Complement activation has been implicated in the pathogenesis of several human diseases. Recently, a monoclonal antibody, (N19-8) that recognizes the human complement protein C5 has been shown to effectively block the cleavage of C5 into C5a and C5b, thereby blocking terminal complement activation. In this study, a recombinant N19-8 scFv antibody fragment was constructed from the N19-8 variable regions, and produced in both mammalian and bacterial cells. The N19-8 scFv bound human C5 and was as potent as the N19-8 monoclonal antibody at inhibiting human C5b-9-mediated hemolysis of chicken erythrocytes. In contrast, the N19-8 scFv only partially retained the ability of the N19-8 monoclonal antibody to inhibit C5a generation. To investigate the ability of the N19-8 scFv to inhibit complement-mediated tissue damage, complement-dependent myocardial injury was induced in isolated mouse hearts by perfusion with Krebs-Henseleit buffer containing 6% human plasma. The perfused hearts sustained extensive deposition of human C3 and C5b-9, resulting in increased coronary artery perfusion pressure, end-diastolic pressure, and a decrease in heart rate until the hearts ceased beating approximately 10 min after addition of plasma. Hearts treated with human plasma supplemented with either the N19-8 monoclonal antibody or the N19-8 scFv did not show any detectable changes in cardiac performance for at least 1 hr following the addition of plasma. Hearts treated with human plasma alone showed extensive deposition of C3 and C5b-9, while hearts treated with human plasma containing N19-8 scFv showed extensive deposition of C3, but no detectable deposition of C5b-9. Administration of a 100 mg bolus dose of N19-8 scFv to rhesus monkeys inhibited the serum hemolytic activity by at least 50% for up to 2 hr. Pharmacokinetic analysis of N19-8 scFv serum levels suggested a two-compartment model with a T1/2 alpha of 27 min. Together, these data suggest the recombinant N19-8 scFv is a potent inhibitor of the terminal complement cascade and may have potential in vivo applications where short duration inhibition of terminal complement activity is desirable.