Infection of swine with virulent porcine reproductive and respiratory syndrome (PRRS) virus induced a rapid, robust antibody response that comprised predominantly nonneutralizing antibodies and waned after approximately 3 months. In contrast, the initial onset of virus-specific interferon (IFN)-gamma-secreting cells (SC) in the pig lymphocyte population remained at a fairly low level during this period and then increased gradually in frequency, plateauing at 6 months postinfection. A similar polarization of the host humoral and cellular immune responses was also observed in pigs immunized with a PRRS-modified live virus (MLV) vaccine. Even coadministration of an adjuvant that enhanced the immune response to a pseudorabies (PR) MLV vaccine failed to alter the induction of PRRS virus-specific IFN-gamma SC (comprising predominantly CD4/CD8 alpha double positive memory T cells with a minority being typical CD4(-)/CD8 alpha beta(+) T cells) and the generation of neutralizing antibodies. Moreover, unlike inactivated PR virus, nonviable PRRS virus did not elicit virus-neutralizing antibody production. Presumably, an intrinsic property of this pathogen delays the development of the host IFN-gamma response and preferentially stimulates the synthesis of antibodies incapable of neutralization.

After infection of swine with porcine reproductive and respiratory syndrome virus (PRRSV), there is a rapid rise of PRRSV-specific nonneutralizing antibodies (NNA), while neutralizing antibodies (NA) are detectable not sooner than 3 weeks later. To characterize neutralizing epitopes, we selected phages from a 12-mer phage display library using anti-PRRSV neutralizing monoclonal antibody (MAb) ISU25-C1. In addition, phages carrying peptides recognized by swine antibodies with high seroneutralizing titer were isolated after subtracting from the library those clones binding to swine anti-PRRSV serum with no neutralizing activity. Two epitopes located in the ectodomain of PRRSV GP5 were identified. One of these epitopes, which we named epitope B, was recognized both by neutralizing MAb ISU25-C1 and swine neutralizing serum (NS) but not by swine nonneutralizing serum (NNS), indicating that it is a neutralizing epitope. Epitope B is sequential, conserved among isolates, and not immunodominant. Antibodies directed against it are detected in serum late after infection. In contrast, the other epitope, which we named epitope A, is hypervariable and immunodominant. Antibodies against it appear early after infection with PRRSV. This epitope is recognized by swine NNA but is not recognized by either neutralizing MAb ISU25-C1 or swine NA, indicating that it is not involved in PRRSV neutralization. During infection with PRRSV, epitope A may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of NA against epitope B for at least 3 weeks. These results are relevant to the design of vaccines against PRRSV.

Immune mechanisms mediating protective immunity against porcine reproductive and respiratory syndrome virus (PRRSV) are not well understood. The PRRSV-specific humoral immune response has been dismissed as being ineffective and perhaps deleterious for the host. The function of PRRSV antibodies in protective immunity against infection with a highly abortifacient strain of this virus was examined by passive transfer experiments in pregnant swine. All of a group of pregnant gilts (n = 6) that received PRRSV immunoglobulin (Ig) from PRRSV-convalescent, hyperimmune animals were fully protected from reproductive failure as judged by 95% viability of offspring at weaning (15 days of age). On the other hand, the totality of animals in a matched control group (n = 6) receiving anti-pseudorabies virus (PRV) Ig exhibited marked reproductive failure with 4% survival at weaning. Besides protecting the pregnant females from clinical reproductive disease, the passive transfer of PRRSV Ig prevented the challenge virus from infecting the dams and precluded its vertical transmission, as evidenced by the complete absence of infectious PRRSV from the tissues of the dams and lack of infection in their offspring. In summary, these results indicate that PRRSV-Igs are capable of conferring protective immunity against PRRSV and furthermore that these Igs can provide sterilizing immunity in vivo.

We studied the persistence of porcine reproductive and respiratory syndrome virus (PRRSV) in individual experimentally infected pigs, during a period of up to 150 days postinfection (dpi). The results of this study suggest that the persistence of PRRSV involves continuous viral replication but that it is not a true steady-state persistent infection. The virus eventually clears the body and seems to do it in most of the animals by 150 dpi or shortly thereafter. High genetic stability was seen for several regions of the persistent PRRSV's genome, although some consistent mutations in the genes of envelope glycoproteins and M protein were also observed.

Understanding the dynamics of porcine reproductive and respiratory syndrome virus (PRRSV) persistence in individual pigs is essential to the development of successful control programs. The objectives of this study were to investigate the proportion of inoculated pigs that become persistently infected with PRRSV and the duration of their infection. Additionally, different diagnostic techniques that detect persistent infections were compared. Twenty-eight 35-day-old pigs were inoculated with PRRSV. Serum and tonsil biopsy samples were collected on days 0, 7, 14, and 28 and then approximately monthly thereafter until day 251 postinoculation (p.i.). Tonsil, lymph node, and lung samples were collected following euthanasia on day 251 p.i. Virus was isolated from serum and tonsil biopsy samples that had been collected through days 28 and 56 p.i., respectively.

The nucleotide sequence of a highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) was determined. Transfection of MARC-145 cells with capped in vitro transcripts derived from a full-length cDNA clone of the viral genome resulted in infectious PRRSV with growth characteristics similar to that of the parental virus. Primer extension analysis revealed that during replication, the viral polymerase corrected the two nonviral guanosine residues present at the 5' terminus of the transfected transcripts. Animal studies showed that the cloned virus induced hyperthermia, persistent viremia, and antibody response, similar to that observed with the parental virus. Contact transmission occurred rapidly within 3 days of introduction of naïve pigs into the group of clone virus-inoculated pigs. These results suggest that the cloned virus retains the in vivo virulence and contagion properties of the parental virus, thus, providing the background for reverse genetics manipulation in systematic examination of attenuation and virulence phenotypes.

Summary. Although live-attenuated vaccines have been used for some time to control clinical symptoms of the porcine reproductive and respiratory syndrome (PRRS), the molecular bases for the attenuated phenotype remain unclear. We had previously determined the genomic sequence of the pathogenic PRRSV 16244B. Limited comparisons of the structural protein coding sequence of an attenuated vaccine strain have shown 98% homology to the pathogenic 16244B. Here we have confirmed the attenuated phenotype and determined the genomic sequence of that attenuated PRRSV vaccine and compared it to its parental VR-2332 and the 16244B strains. The attenuated vaccine sequence was colinear with that of the strain 16244B sequence containing no gaps and 212 substitutions over 15,374 determined nucleotide sequence. We identified nine amino acid changes distributed in Nsp1␤, Nsp2, Nsp10, ORF2, ORF3, ORF5 and ORF6. These changes may provide the molecular bases for the observed attenuated phenotype.

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