The porcine humoral immune response against pseudorabies virus specifically targets attachment sites on glycoprotein gC

High titers of virus-neutralizing antibodies directed against glycoprotein gC of Pseudorabies virus (PRV) (Suid herpesvirus 1) are generally observed in the serum of immunized pigs. A known function of the glycoprotein gC is to mediate attachment of PRV to target cells through distinct viral heparin-binding domains (HBDs). Therefore, it was suggested that the virus-neutralizing activity of anti-PRV sera is directed against HBDs on gC. To address this issue, sera with high virus-neutralizing activity against gC were used to characterize the anti-gC response. Epitope mapping demonstrated that amino acids of HBDs are part of an antigenic antibody binding domain which is located in the N-terminal part of gC. Binding of antibodies to this antigenic domain of gC was further shown to interfere with the viral attachment. Therefore, these results show that the viral HBDs are accessible targets for the humoral anti-PRV response even after tolerance induction against self-proteins, which utilize similar HBDs to promote host protein-protein interactions. The findings indicate that the host's immune system can specifically block the attachment function of PRV gC. Since HBDs promote the attachment of a number of herpesviruses, the design of future antiherpesvirus vaccines should aim to induce a humoral immune response that prevents HBD-mediated viral attachment.     

CD8 T cells require gamma interferon to clear borna disease virus from the brain and prevent immune system-mediated neuronal damage

Borna disease virus (BDV) frequently causes meningoencephalitis and fatal neurological disease in young but not old mice of strain MRL. Disease does not result from the virus-induced destruction of infected neurons. Rather, it is mediated by H-2(k)-restricted antiviral CD8 T cells that recognize a peptide derived from the BDV nucleoprotein N. Persistent BDV infection in mice is not spontaneously cleared. We report here that N-specific vaccination can protect wild-type MRL mice but not mutant MRL mice lacking gamma interferon (IFN-gamma) from persistent infection with BDV. Furthermore, we observed a significant degree of resistance of old MRL mice to persistent BDV infection that depended on the presence of CD8 T cells. We found that virus initially infected hippocampal neurons around 2 weeks after intracerebral infection but was eventually cleared in most wild-type MRL mice. Unexpectedly, young as well as old IFN-gamma-deficient MRL mice were completely susceptible to infection with BDV. Moreover, neurons in the CA1 region of the hippocampus were severely damaged in most diseased IFN-gamma-deficient mice but not in wild-type mice. Furthermore, large numbers of eosinophils were present in the inflamed brains of IFN-gamma-deficient mice but not in those of wild-type mice, presumably because of increased intracerebral synthesis of interleukin-13 and the chemokines CCL1 and CCL11, which can attract eosinophils. These results demonstrate that IFN-gamma plays a central role in host resistance against infection of the central nervous system with BDV and in clearance of BDV from neurons. They further indicate that IFN-gamma may function as a neuroprotective factor that can limit the loss of neurons in the course of antiviral immune responses in the brain.     

Mapping of the structural gene of pseudorabies virus glycoprotein A and identification of two non-glycosylated precursor polypeptides.

Cell-free translation of pseudorabies virus RNA isolated during the late phase of the infectious cycle yielded a variety of polypeptides. A monoclonal antibody directed against one of the major viral glycoproteins, gA, immunoprecipitated two polypeptides ranging in molecular weight from 78K to 83K. To localize the structural gene for gA, we used cloned BamHI fragments of the viral DNA to select specific mRNA species and immunoprecipitated their in vitro translation products with the anti-gA monoclonal antibody. This allowed us to map the genomic region encoding the mRNA for the gA within the short unique region of the viral genome on BamHI fragments 7 and 12. Additional polypeptides encoded by this region were characterized by their electrophoretic mobility. In three virus strains tested a similar, but strain-specific, pattern of the two gA precursors was found which was not dependent on the host cell or the state of infection after reaching the late phase.     

Demonstration of three major species of pseudorabies virus glycoproteins and identification of a disulfide-linked glycoprotein complex.

The glycoproteins of pseudorabies virus (PRV) Phylaxia were characterized with monoclonal antibodies as specific reagents. Three major structural glycoproteins with molecular weights of 155,000 (155K) (gC), 122K (gA), and 90K (gB) could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We investigated the processing of glycoproteins gA, gB, and gC by in vitro translation, pulse-chase experiments, and in the presence of the ionophore monensin which inhibits glycosylation. gA and gB were found to compose a single polypeptide, whereas gC was found to be a disulfide-linked glycoprotein complex. Immunoprecipitates formed with the aid of anti-gC monoclonal antibodies gave rise to three glycoprotein bands (gC0 [120K], gC1 [67K], and gC2 [58K]) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Limited proteolysis of gC0, gC1, and gC2 resulted in peptide maps of gC0 related to those of both gC1 and gC2. No common peptide bands between gC1 and gC2, however, were seen. We suggest that (i) gC1 and gC2 arise by proteolytic cleavage from the same precursor molecule and stay joined via disulfide bridges and (ii) gC0 is an uncleaved precursor.     

Prevention of virus persistence and protection against immunopathology after Borna disease virus infection of the brain by a novel Orf virus recombinant

The Parapoxvirus Orf virus represents a promising candidate for novel vector vaccines due to its immune modulating properties even in nonpermissive hosts such as mouse or rat. The highly attenuated Orf virus strain D1701 was used to generate a recombinant virus (D1701-VrVp40) expressing nucleoprotein p40 of Borna disease virus, which represents a major antigen for the induction of a Borna disease virus-specific humoral and cellular immune response. Infection with Borna disease virus leads to distinct neurological symptoms mediated by the invasion of activated specific CD8+ T cells into the infected brain. Usually, Borna disease virus is not cleared from the brain but rather persists in neural cells. In the present study we show for the first time that intramuscular application of the D1701-VrVp40 recombinant protected rats against Borna disease, and importantly, virus clearance from the infected brain was demonstrated in immunized animals. Even 4 and 8 months after the last immunization, all immunized animals were still protected against the disease. Initial characterization of the immune cells attracted to the infected brain areas suggested that D1701-VrVp40 mediated induction of B cells and antibody-producing plasma cells as well as T cells. These findings suggest the induction of various defense mechanisms against Borna disease virus. First studies on the role of antiviral cytokines indicated that D1701-VrVp40 immunization did not lead to an enhanced early response of gamma or alpha interferon or tumor necrosis factor alpha. Collectively, this study describes the potential of the Orf virus vector system in mediating long-lasting, protective antiviral immunity and eliminating this persistent virus infection without provoking massive neuronal damage.     

Appearance of the bona fide spiral tubule of ORF virus is dependent on an intact 10-kilodalton viral protein

Parapoxviruses can be morphologically distinguished from other poxviruses in conventional negative staining electron microscopy (EM) by their ovoid appearance and the spiral tubule surrounding the virion's surface. However, this technique may introduce artifacts. We have examined Orf virus (ORFV; the prototype species of the Parapoxvirus genus) by cryoelectron microscopy (cryo-EM) and cryo-negative staining EM. From these studies we suggest that the shape and unique spiral tubule are authentic features of the parapoxviruses. We also constructed an ORFV mutant deleted of a gene encoding a 10-kDa protein, which is an orthologue of the vaccinia virus (VACV) 14-kDa fusion protein, and investigated its ultrastructure. This mutant virus multiplied slowly in permissive cells and produced infectious but morphologically aberrant particles. Mutant virions lacked the spiral tubule but displayed short disorganized tubules similar to those observed on the surface of VACV. In addition, thin extensions or loop-like structures were appended to the ORFV mutant particles. We suggest that these appended structures arise from a failure of the mutant virus particles to properly seal and that the sealing activity is dependent on the 10-kDa protein.     

Specific pseudorabies virus infection of the rat visual system requires both gI and gp63 glycoproteins.

Transneuronal transport of pseudorabies virus (PRV) from the retina to visual centers that mediate visual discrimination and reflexes requires specific genes in the unique short region of the PRV genome. In contrast, these same viral genes are not required to infect retinorecipient areas of the brain involved in circadian rhythm regulation. In this report, we demonstrate that viral mutants carrying defined deletions of the genes encoding glycoprotein gI or gp63, or both, result in the same dramatic transport defect. Efficient export of either gI or gp63 from the endoplasmic reticulum to the Golgi apparatus in a fibroblast cell line requires the presence of both proteins. We also show that gI and gp63 physically interact, as demonstrated by pulse-chase and sucrose gradient sedimentation experiments. Complex formation is rapid compared with homodimerization of PRV glycoprotein gII. We suggest that gI and gp63 function in concert to affect neurotropism in the rat visual circuitry and that a heterodimer is likely to be the unit of function.