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Salivary excretion of rabies virus was evaluated in 14 adult vampire bats (Desmodus rotundus) intramuscularly injected with a large dose (106 MICLD50) of vampire rabies virus variant CASS88. Saliva samples were obtained from surviving bats every other day for 30 days, then weekly for 2 months, and finally 1 and 2 years later. Rabies virus was isolated in murine neuroblastoma cells and in randomly selected cases by PCR. Rabies virus was not detected in the saliva of any of the 11 animals that succumbed (somewhat early) to rabies challenge, nor in the control bats. In contrast, virus was detected early, and only once (days 6, 6 and 21) in each of the three animals that survived rabies challenge and remained healthy for at least 2 years after challenge. At that time even vigorous dexamethasone and cyclosporine administration failed to provoke further viral excretion.

RABIES infection of domestic and wild animals is a serious problem throughout the world. The major disease vector in Europe is the red fox ( Vulpes vulpes ) and rabies control has focused on vaccinating and/or culling foxes. Culling has not been effective, and the distribution of live vaccine baits is the only appropriate method for the vaccination of wild foxes 1 . Although some European countries have conducted field vaccination campaigns using attenuated rabies virus strains 2-5 , their use has not been extensively approved because they retain pathogenicity for rodents and can revert to virulence 6,7 . These strains cannot be used in North America because they are pathogenic for the striped skunk( Mephitis mephitis ) 8 and are ineffective in the racoon ( Procyonlotor ) 9 . We have constructed a recombinant vaccinia virus10, VVTGgRAB, expressing the surface glycoprotein (G) of rabies virus (ERA strain) 11–13 . The recombinant was a highly effective vaccine in experimental animals 11–13 , in captive foxes 14,15 and in racoons 9 . We report here the results of a large-scale campaign of fox vaccination in a 2,200 km 2 region of southern Belgium, an area in which rabies is prevalent. After distribution, 81% of foxes inspected were positive for tetracycline, a biomarker included in the vaccine bait and, other than one rabid fox detected close to the periphery of the treated area, no case of rabies, either in foxes or in domestic livestock, has been reported in the area.

To improve both safety and stability of the oral vaccines used in the field to vaccinate foxes against rabies, a recombinant vaccinia virus, which expresses the immunizing G protein of rabies virus has been developed by inserting the cDNA which codes for the immunogenic glycoprotein of rabies virus into the thymidine kinase (TK) gene of the Copenhagen strain of vaccinia virus. The efficacy of this vaccine was tested by the oral route, primarily in foxes. The immunity conferred, a minimum of 12 months in cubs and 18 months in adult animals, corresponds to the duration of the protection required for vaccination of foxes in the field. Innocuity was tested in foxes, domestic animals, and in numerous European wild animal species that could compete with the red fox for the vaccine bait. No clinical signs or lesions were observed in any of the vaccinated animals during a minimum of 28 days post vaccination. Moreover, no transmission of immunizing doses of the recombinant occurred between foxes or other species tested. To study the stability of the vaccine strain, baits containing the vaccine were placed in the field. Despite considerable variations of environmental temperatures, the vaccine remained stable for at least one month. Because bait is taken within one month, it can be assumed that most animals taking the baits are effectively vaccinated. To test the field efficacy of the recombinant vaccine, large-scale campaigns of fox vaccination were set up in a 2200 km2 region of southern Belgium, were rabies was prevalent. A dramatic decrease in the incidence of rabies was noted after the campaigns. The recombinant is presently used to control wildlife rabies in the field both in several European countries and in the United States.

An infectious cDNA clone of the hypervirulent bovine viral diarrhoea virus (BVDV) strain 890 (isolate 256) was produced by a streamlined PCR procedure. As compared to the published sequence of strain 890, the nucleotide sequencing of cloned cDNA corresponding to isolate 256 revealed several mutations seven of which were attributed to the cloning procedure. The infectious transcript was transfected into permissive cells and led to viral multiplication ( AvrII+ strain). In vitro, viral titres reached by the parental strain exceed those of the AvrII+ strain by more than one order of magnitude. The latter was clearly less virulent to young calves as indicated by clinical, haematological and virological parameters. Thirty-four days after inoculation with AvrII+ strain, calves were challenged with the virulent parental strain. The animals were protected as compared to unvaccinated controls. Therefore, our approach led to the production of an attenuated strain with potential use as a vaccine strain and will be useful for studies of virulence determinants in BVDV-2.

Neospora caninum and Toxoplasma gondii are closely related protozoan parasites. The latter, which was first described in 1908, has a two stage asexual lifecycle with tachyzoites multiplying during an acute infection and bradyzoites in tissue cysts in the persistent form of the infection. Its full lifecycle, however, involving the production of oocysts by Felidae was not established until 1970. N caninum, on the other hand, was first described in 1984 when it was shown to be the cause of death in a group of puppies and was named in 1988. To date it is known to exist as bradyzoites in tissue cysts, and as tachyzoites; a sexual lifecycle involving the production of oocysts has not been demonstrated, although it has been predicted that there is likely to be a definitive carnivore host with a role similar to the cat in toxoplasmosis. To study the distribution of N caninum in the environment sera from red foxes (Vulpes vulpes) caught in Belgium, which were sampled as part of the rabies control programme, were examined for evidence of antibodies to N caninum and T gondii.

A new genotype of bovine viral diarrhoea virus (BvDv), designated BVDV-2, has emerged in the last decade and in recent years the prevalence of BVDV-2 strains has increased. A vaccination-challenge study was carried out to determine the cross-protective efficacy of a commercial inactivated vaccine containing a BVDV-1 strain. A group of five BVDv-free calves was vaccinated twice and a second group of five calves served as negative controls. Two months after the first vaccination, all the calves were challenged intranasally with BVDV-2 strain BVD890. The clinical signs of disease, the changes in haematological variables and the level of viraemia were significantly less in the vaccinated group.

Several fox vaccination campaigns against rabies have been undertaken in Belgium by using a vaccinia-rabies recombinant virus distributed in baits in the field. However, foxes and other wild animals that may ingest the baits could be infected at the same time by another orthopoxvirus, such as cowpox virus, which circulates in wildlife. Recombination between the two viruses could therefore occur. A serological survey for antibodies to orthopoxvirus, and particularly to cowpox virus, was undertaken in foxes and in several other wild species. Antibodies were detected only in two rodent species, in 16 of 25 bank voles (64 per cent) and in two of 29 woodmice (7 per cent). The risk of virus recombination in wildlife can therefore be considered to be extremely low.

The nucleotide sequence of a 10.5 kb region (map position 0.332 to 0.410) of bovine herpesvirus type 1 (BHV-1) was determined. This region contained three open reading frames (ORFs) homologous to herpes simplex virus DNA polymerase catalytic subunit (DNApol, UL30), major DNA-binding protein (MDBP, UL29) and ICP18.5 assembly protein (ICP18.5, UL28). The BHV-1 DNApol, MDBP and ICP18.5 ORFs were 1 246, 1 203 and 826 amino acids long with a calculated molecular mass of 134.2 kDa, 124.4 kDa and 86.9 kDa, respectively. They showed a high homology with alphaherpesvirus homologs despite large differences in the G + C content of the UL30-UL28 segment ranging from 44.4% for varicella zoster virus to 71.5% for BHV-1. Particularly well conserved among Alphaherpesvirinae are the putative functional domains of the DNApol and MDBP proteins which are discussed. Phylogenetic analysis revealed that BHV-1 clustered in the Varicellovirus genus with the animal D-type viruses. In this group, the BHV-1 position was shown to vary according to the investigated genes. Indeed, pseudorabies virus clustered with BHV-1 in the DNApol tree but with equine herpesvirus 1 in the ICP18.5 tree.

In the epidemiology of bovine viral diarrhoea (BVD), immunotolerant – persistently infected animals (IPI) appear to be major sources of contamination. These animals produce large quantities of replicating virus and have therefore been proposed as being responsible for generating antigenic variability. However, limited studies have failed to detect antigenic or genetic changes in viruses isolated at different times from IPI. An hypothesis is that the immunotolerance of IPI against their homologous strain is accompanied by immune elimination of antigenic variants. The presence of an IPI in a herd could therefore limit antigenic variation, eventually leading to the existence of herd specific strains. To verify this hypothesis we characterized, against a panel of monoclonal antibodies, 37 BVD virus strains isolated from IPI of 12 herds in Eastern Belgium. Intra-herd antigenic variation was compared to inter-herd variation. Antigenic variation within herds was found to be surprisingly high but, nevertheless, significantly lower than variation between herds.