R.J.M. Moormann

Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus

Abstract The genome of Lelystad virus (LV), a positive-strand RNA virus, is 15 kb in length and contains 8 open reading frames (ORFs) that encode putative viral proteins. ORFs 2 to 7 were cloned in plasmids downstream of the Sp6 RNA polymerase promoter, and the translation of transcripts generated in vitro yielded proteins that could be immunoprecipitated with porcine anti-LV serum. Synthetic polypeptides of 15 to 17 amino acids were selected from the amino acid sequences of ORFs 2 to 7 and antipeptide sera were raised in rabbits. Antisera that immunoprecipitated the in vitro translation products of ORFs 2 to 5 and 7 were obtained. Sera containing antibodies directed against peptides from ORFs 3 to 7 reacted positively with LV-infected alveolar lung macrophages in the immunoperoxidase monolayer assay. Using these antipeptide sera and porcine anti-LV serum, we identified three structural proteins and assigned their corresponding genes. Virions were found to contain a nucleocapsid protein of 15 kDa (N), an unglycosylated membrane protein of 18 kDa (M), and a glycosylated membrane protein of 25 kDa (E). The N protein is encoded by ORF7, the M protein is encoded by ORF6, and the E protein is encoded by ORF5. The E protein in virus particles contains one or two N-glycans that are resistant to endo-β-N-acetyl-d-glucosaminidase H. This finding indicates that the high-mannose glycans are processed into complex glycans in the Golgi compartment. The protein composition of the LV virions further confirms that LV is evolutionarily related to equine arteritis virus, simian hemorrhagic fever virus, and lactate dehydrogenase-elevating virus.

Lelystad virus, the cause of porcine epidemic abortion and respiratory syndrome: a review of mystery swine disease research at Lelystad

This paper reviews the laboratory investigations that led us to isolate the Lelystad virus and demonstrate that this virus causes mystery swine disease. We describe: 1) isolating the virus from the disease; 2) characterizing the virus as a new enveloped RNA virus; 3) reproducing the disease experimentally with the isolated Lelystad virus; 4) isolating the virus from the experimentally induced disease.

Marker vaccines, virus protein-specific antibody assays and the control of Aujeszky's disease

Vaccination of pigs is widely practised to control Aujeszkys disease (AD). Molecular biological research revealed that several conventionally attenuated virus vaccines harbour deletions in their genomes. The deleted genes are nonessential for virus replication and can be involved in the expression of virulence. These findings have prompted several groups to construct well-characterized deletion mutants of AD virus that do not express either glycoprotein gI, gX or gIII. These mutants have also been rendered thymidine kinase negative. Although data on vaccine efficacy and safety have been published, widely varying test conditions have made it impossible to identify the most efficacious deletion mutant vaccine(s). Vaccination enhances the amount of virus required for infection and reduces, but does not prevent, the shedding of virulent virus and the establishment of latency in pigs infected with virulent AD virus. Therefore, while a vaccination programme will reduce the circulation of virus in the field, it will not eliminate AD virus from pig populations. To eradicate AD, the ability to differentiate infected from vaccinated pigs is crucial. The use of marker vaccines enables us to identify infected pigs in vaccinated populations by detecting antibodies against the protein whose gene is deleted from vaccine strains. The antibody response to gI appears to persist for more than 2 years, and all of about 300 field strains tested so far express gI. The use of vaccines lacking gI in combination with an enzyme linked immunosorbent assay to detect antibodies to gI and culling of gI-seropositive pigs, may help to eradicate AD in countries where vaccination is widely practised.

Inactivation of the thymidine kinase gene of a gI deletion mutant of pseudorabies virus generates a safe but still highly immunogenic vaccine strain.

In an earlier report, we described the construction of the genetically engineered pseudorabies virus strain 2.4N3A which does not express glycoprotein gI. Although this strain showed a strongly reduced virulence in 10-week-old seronegative pigs, it could still cause severe disease or death in 3-day-old piglets. To attenuate the strain further, we constructed mutants with a deletion in the viral thymidine kinase gene. One mutant strain, designated 783, has a deletion of 19 base pairs and was shown to be highly immunogenic and safe for vaccination of pigs against pseudorabies virus.

Rift Valley fever virus subunit vaccines confer complete protection against a lethal virus challenge

Rift Valley fever virus (RVFV) is an emerging mosquito-borne virus causing significant morbidity and mortality in livestock and humans. Rift Valley fever is endemic in Africa, but also outside this continent outbreaks have been reported. Here we report the evaluation of two vaccine candidates based on the viral Gn and Gc envelope glycoproteins, both produced in a Drosophila insect cell expression system. Virus-like particles (VLPs) were generated by merely expressing the Gn and Gc glycoproteins. In addition, a soluble form of the Gn ectodomain was expressed and affinity-purified from the insect cell culture supernatant. Both vaccine candidates fully protected mice from a lethal challenge with RVFV. Importantly, absence of the nucleocapsid protein in either vaccine candidate facilitates the differentiation between infected and vaccinated animals using a commercial recombinant nucleocapsid protein-based indirect ELISA.

Heparan sulfate facilitates Rift Valley fever virus entry into the cell

ABSTRACT Rift Valley fever virus (RVFV), an emerging arthropod-borne pathogen, has a broad host and cell tropism. Here we report that the glycosaminoglycan heparan sulfate, abundantly present on the surface of most animal cells, is required for efficient entry of RVFV. Entry was significantly reduced by preincubating the virus inoculum with highly sulfated heparin, by enzymatic removal of heparan sulfate from cells and in cells genetically deficient in heparan sulfate synthesis.

Acid-activated structural reorganization of the Rift Valley fever virus Gc fusion protein

ABSTRACT The entry of the enveloped Rift Valley fever virus (RVFV) into its host cell is mediated by the viral glycoproteins Gn and Gc. We investigated the RVFV entry process and, in particular, its pH-dependent activation mechanism using our recently developed nonspreading-RVFV-particle system. Entry of the virus into the host cell was efficiently inhibited by lysosomotropic agents that prevent endosomal acidification and by compounds that interfere with dynamin- and clathrin-dependent endocytosis. Exposure of plasma membrane-bound virions to an acidic pH (<pH 6) equivalent to the pH of late endolysosomal compartments allowed the virus to bypass the endosomal route of infection. Acid exposure of virions in the absence of target membranes triggered the class II-like Gc fusion protein to form extremely stable oligomers that were resistant to SDS and temperature dissociation and concomitantly compromised virus infectivity. By targeted mutagenesis of conserved histidines in Gn and Gc, we demonstrated that mutation of a single histidine (H857) in Gc completely abrogated virus entry, as well as acid-induced Gc oligomerization. In conclusion, our data suggest that after endocytic uptake, RVFV traffics to the acidic late endolysosomal compartments, where histidine protonation drives the reorganization of the Gc fusion protein that leads to membrane fusion.

Comparison of the protective efficacy of recombinant pseudorabies viruses against pseudorabies and classical swine fever in pigs; influence of different promoters on gene expression and on protection

The glycoprotein E (gE) locus in the genome of pseudorabies virus (PRV) was used as an insertion site for the expression of glycoprotein E1 of classical swine fever virus (CSFV). Transcription of E1 in the recombinants M401, M402 or M403 was regulated by the gD promoter of PRV, the immediate early gene promoter of human cytomegalovirus, or the gE promoter of PRV, respectively. Groups of four pigs were vaccinated once intramuscularly with 10(6) plaque forming units (p.f.u.) of the recombinant viruses and challenged intranasally with 100 50% lethal doses of virulent CSFV and with 10(5) p.f.u. of virulent PRV. All pigs vaccinated with M402 were fully protected against both classical swine fever and pseudorabies.

Rift Valley Fever Vaccine Development, Progress and Constraints

The workshop Rift Valley Fever Vaccine Development, Progress and Constraints was organized by the Food and Agriculture Organization of the United Nations (FAO) and the Central Veterinary Institute of Wageningen University and Research Centre, under the umbrella of the Global Framework for the Progressive Control of Transboundary Animal Diseases, a joint initiative of FAO and the World Organisation for Animal Health. The workshop was supported by the Netherlands Ministry of Economic Affairs, Agriculture and Innovation, and by the US Centers for Disease Control and Prevention; other participants included the World Health Organization and the International Atomic Energy Agency. The meeting occurred January 19–21, 2011, at FAO headquarters in Rome, Italy, and was attended by 34 leading scientists in Rift Valley fever virus (RVFV) vaccine development, representatives of international organizations, and policy makers. Stakeholders from industry were represented by the International Federation for Animal Health. The main objective of the meeting was to gain consensus about desired characteristics of novel veterinary RVFV vaccines and to discuss how incentives can be established to ensure that these vaccines come to market.

A Single Vaccination with an Improved Nonspreading Rift Valley Fever Virus Vaccine Provides Sterile Immunity in Lambs

Rift Valley fever virus (RVFV) is an important pathogen that affects ruminants and humans. Recently we developed a vaccine based on nonspreading RVFV (NSR) and showed that a single vaccination with this vaccine protects lambs from viremia and clinical signs. However, low levels of viral RNA were detected in the blood of vaccinated lambs shortly after challenge infection. These low levels of virus, when present in a pregnant ewe, could potentially infect the highly susceptible fetus. We therefore aimed to further improve the efficacy of the NSR vaccine. Here we report the expression of Gn, the major immunogenic protein of the virus, from the NSR genome. The resulting NSR-Gn vaccine was shown to elicit superior CD8 and CD4-restricted memory responses and improved virus neutralization titers in mice. A dose titration study in lambs revealed that the highest vaccination dose of 106.3 TCID50/ml protected all lambs from clinical signs and viremia. The lambs developed neutralizing antibodies within three weeks after vaccination and no anamnestic responses were observed following challenge. The combined results suggest that sterile immunity was achieved by a single vaccination with the NSR-Gn vaccine.