F.A.M. Rijsewijk

Envelope Protein Requirements for the Assembly of Infectious Virions of Porcine Reproductive and Respiratory Syndrome Virus

ABSTRACT Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2a, E, GP3, and GP4. Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indicating that these proteins interact with each other and are assembled into virions as a multimeric complex. Independent evidence for such complexes was obtained by coexpression of the minor envelope proteins in BHK-21 cells using a Semliki Forest virus expression system. By analyzing the maturation of their N-linked oligosaccharides, we found that the glycoproteins were each retained in the endoplasmic reticulum unless expressed together, in which case they were collectively transported through the Golgi complex to the plasma membrane and were even detected in the extracellular medium. As the PRRSV particles lacking the minor envelope proteins are not infectious, we hypothesize that the virion surface structures formed by these proteins function in viral entry by mediating receptor binding and/or virus-cell fusion.

A conventionally attenuated glycoprotein E-negative strain of bovine herpesvirus type 1 is an efficacious and safe vaccine.

We examined the vaccine properties of a conventionally attenuated bovine herpesvirus type 1 (BHV-1) mutant strain A. This strain has a deletion that encompasses the glycoprotein E gene and may therefore be used as a marker vaccine. We compared strain A with a commercial live BHV-1 vaccine B. Calves were intranasally vaccinated with strain A or with vaccine B. Strain A was virtually avirulent for young calves. After challenge, vaccinated calves were protected against disease and virus shedding was considerably reduced. After dexamethasone treatment, strain A was not recovered, whereas vaccine B and challenge virus were. We conclude that strain A is suitable for inclusion in a vaccine and has the potential advantage of being used as a marker vaccine.

A glycoprotein E deletion mutant of bovine herpesvirus 1 is avirulent in calves

A marker vaccine elicits an antibody response in the host that can be distinguished from the antibody response induced by a wild-type strain. To obtain a bovine herpesvirus 1 (BHV-1) marker vaccine, we constructed a glycoprotein E (gE) deletion mutant. This was obtained by removing the complete gE coding region from the BHV-1 genome. To attenuate the gE deletion mutant further, we also introduced a small deletion in the thymidine kinase (TK) gene. We selected three mutants: the gE deletion mutant, a TK deletion mutant and a gE/TK double deletion mutant, and examined their virulence and immunogenicity in calves. After intranasal inoculation, the TK deletion mutant showed some residual virulence, whereas the gE and gE/TK deletion mutants were avirulent. The calves inoculated with the deletion mutants were protected against disease after challenge exposure and shed significantly less virus than control calves. Deleting the gE gene, therefore, has little effect on the immunogenicity of BHV-1, but is sufficient to reduce the virulence of BHV-1 in calves. These findings led us to conclude that the gE deletion mutant is a good candidate for a modified live BHV-1 marker vaccine.

Virulence, immunogenicity and reactivation of bovine herpesvirus 1 mutants with a deletion in the gC, gG, gI, gE, or in both the gI and gE gene

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.

Advances in the development and evaluation of bovine herpesvirus 1 vaccines.

This review deals with conventional and modern bovine herpesvirus 1 (BHV1) vaccines. Conventional vaccines are widely used to prevent clinical signs of infectious bovine rhinotracheitis. The use of conventional vaccines, however, does not appear to have resulted in reduction of the prevalence of infection. Novel BHV1 marker vaccines comprise either mutants with a deletion in one of the non-essential genes, or subunit vaccines that contain one or more glycoproteins. These marker vaccines can be used in conjunction with companion diagnostic tests to differentiate between infected and vaccinated cattle. Their efficacy has been evaluated in vaccination-challenge experiments, transmission experiments and in field trials. The results demonstrate that the marker vaccines can contribute to the eventual eradication of BHV1. However, there remains room for improvement of BHV1 marker vaccines.

An inactivated vaccine based on a glycoprotein E-negative strain of bovine herpesvirus 1 induces protective immunity and allows serological differentiation

The bovine herpesvirus 1 (BHV1) strain Za is a conventionally attenuated strain with a 2.7 kb deletion that encompasses the complete coding region for glycoprotein E (gE). This gE-negative strain was used as whole-virus antigen in an inactivated virus vaccine. Three different antigen concentrations of this vaccine were evaluated for safety and efficacy in a vaccination-challenge experiment in calves. No adverse effects were observed in any of the calves vaccinated with the gE-negative vaccines. Calves given the vaccine with the highest antigen concentration were adequately protected against challenge; clinical symptoms were virtually absent and challenge virus shedding was significantly reduced as compared with unvaccinated calves. We developed a sensitive blocking enzyme-linked immunosorbent assay (ELISA) to detect antibodies against gE. After vaccination, calves did not produce antibodies against gE, but these antibodies were detectable within 2 weeks after challenge both in vaccinated and in unvaccinated calves. These results demonstrate the efficacy of a gE-negative inactivated BHV1 vaccine and the detectability of antibodies against gE after infection. The combined use of the marker vaccine and the gE-blocking ELISA makes it possible to differentiate between vaccinated animals and infected animals. This possibility may be very useful in BHV1 control programmes.

An enzyme-linked immunosorbent assay to detect antibodies against glycoprotein gE of bovine herpesvirus 1 allows differentiation between infected and vaccinated cattle

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against glycoprotein gE (gE) of bovine herpesvirus 1 (BHV1). The assay is based on the use of two monoclonal antibodies directed against different antigenic domains on gE. Sera from uninfected cattle and cattle that had been repeatedly vaccinated with gE-negative marker vaccines scored negative, whereas sera from cattle naturally or experimentally infected with BHV1 field strains scored positive in the gE-ELISA. Antibodies against gE appeared in the serum around 11 days after infection. Cattle that were first vaccinated and then challenged, thus having less virus replication, also became gE-seropositive. The sensitivity and specificity of the gE-ELISA is high, and therefore the gE-ELISA is suitable for differentiating between infected cattle and vaccinated cattle with a gE-negative vaccine.

The UL41-encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of MHC class I molecules by bovine herpesvirus 1

The virion host shutoff (vhs) protein of alphaherpesviruses causes a rapid shutoff of host cell protein synthesis. We constructed a bovine herpesvirus 1 (BHV1) deletion mutant in which the putative vhs gene, UL41, has been disrupted. Whereas protein synthesis is inhibited within 3 h after infection with wild-type BHV1, no inhibition was observed after infection with the BHV1(vhs-) deletion mutant. These results indicate that the BHV1 UL41 gene product is both necessary and sufficient for shutoff of host cell protein synthesis at early times post-infection. Using the vhs deletion mutant, we investigated the mechanism of BHV1-induced down-regulation of MHC class I cell surface expression. In contrast to BHV1 wild-type infection, the BHV1(vhs-) mutant allows detection of MHC class I molecules at much later time-points after infection. This illustrates the role the vhs protein plays in MHC class I down-regulation. However, even after infection with BHV1(vhs-), MHC class I cell surface expression is impaired. In BHV1(vhs-)-infected cells, MHC class I molecules are retained within the endoplasmic reticulum (ER). Moreover, the transporter associated with antigen presentation (TAP) is still blocked. Temporal control of viral protein expression using chemical inhibitors shows that viral protein(s) expressed within the early phase of BHV1 infection are responsible for ER retention of MHC class I molecules. These results indicate that multiple mechanisms are responsible for down-regulation of MHC class I molecules in BHV1-infected cells.

The use of marker vaccines in eradication of herpesviruses

Marker vaccines are vaccines that allow serological differentiation between infected and vaccinated individuals. This differentiation is based on the absence of one or more microbial proteins in the vaccine that are present in the wild-type micro-organism. Consequently, after infection, but not after vaccination, an antibody response against that specific protein(s) can be detected. With a protein-specific antibody test infected individuals can thus be distinguished from vaccinated individuals. Marker vaccines against pseudorabies virus (PRV) and against bovine herpesvirus 1 (BHV1) infections have been developed, along conventional routes and by recombinant DNA technology. These vaccines have been shown to be efficacious in reducing (a) clinical signs after infection, (b) wild-type virus replication after infection, and (c) transmission of wild-type virus in the laboratory and in the field. At present, PRV vaccines that lack the gene for the glycoprotein gE are used worldwide in novel eradication programmes. The first phase of such a programme consists of systematic vaccination of pigs on a farm, in a region or an entire country. Experiences in the Netherlands show that it is feasible to eradicate PRV by the intensive use of marker vaccines. Whether, this also holds true for BHV1 is now under investigation.

Immunization of cattle with a BHV1 vector vaccine or a DNA vaccine both coding for the G protein of BRSV

A gE-negative bovine herpesvirus 1 (BHV1) vector vaccine carrying a gene coding for the G protein of bovine respiratory syncytial virus (BRSV) (BHV1/BRSV-G) induced the same high degree of protection in calves against BRSV infection and BHV1 infection as a multivalent commercial vaccine. A DNA plasmid vaccine, carrying the same gene as the BHV1/BRSV-G vaccine, significantly reduced BRSV shedding after BRSV infection compared with that in control calves, but less well than the BHV1/BRSV-G vaccine. Flow cytometric analysis showed a significant relative increase of gamma/delta+ T cells in peripheral blood after BRSV challenge-infection of the calves of the control group but not in the vaccinated groups. These results indicate that the G protein of BRSV can induce significant protection against BRSV infection in cattle, and that the BHV1/BRSV-G vaccine protects effectively against a subsequent BRSV and BHV1 infection.