Evaluation of humoral response and protective efficacy of two inactivated vaccines against bluetongue virus after vaccination of goats.

Bluetongue serotype 8 has become a major animal health issue in the European Union and the European member States have agreed on a vaccination strategy, which involves only inactivated vaccines. In this study, the efficacy of two inactivated vaccines against bluetongue virus serotype 8 (BTV-8) used in Europe since 2008, BTVPUR ALSAP(®) 8 (MERIAL) and BOVILIS(®) BTV8 (Intervet/SP-AH), was evaluated in goats immunized and challenged with BTV-8 field isolates under experimental conditions. Serological, virological and clinical examinations were conducted before and after challenge. Three groups of 10 goats each (groups A, B and C) were randomly constituted and 2 groups (A and C) were subcutaneously vaccinated twice with one dose of the two commercial vaccines BTVPUR ALSAP 8 (group A) or BOVILIS BTV8 (group C) respectively. Animals of the groups A, C and B (B: controls) were challenged with a virulent inoculum containing BTV-8. During the experiment, it was found out that the BTV-8 challenge inoculum was contaminated with another BTV serotype. However, results demonstrated that vaccination of goats with two injections of BTVPUR ALSAP 8 or BOVILIS BTV8 provided a significant clinical protection against a BTV-8 challenge and completely prevented BTV-8 viraemia in all vaccinated animals. Qualitative data showed no difference in the kinetics and levels of the humoral response induced by these two inactivated vaccines.

Use of inactivated bluetongue virus serotype 8 vaccine against virulent challenge in sheep and cattle.

The immunisation properties of an inactivated bluetongue virus serotype 8 (BTV-8) vaccine were evaluated in sheep and cattle. Five sheep were vaccinated with one dose of vaccine and five cattle were vaccinated with two doses 28 days apart. Six sheep and five cattle served as unvaccinated controls. All animals were subjected to a virulent BTV-8 challenge, and safety and antibody responses were monitored. All control animals developed disease and viraemia, while vaccinated animals were clinically protected and viraemia was completely prevented.

Protective duration of immunity of an inactivated bluetongue (BTV) serotype 2 vaccine against a virulent BTV serotype 2 challenge in sheep.

The protective properties of an inactivated bluetongue virus serotype 2 (BTV-2) vaccine were evaluated in sheep. Sheep (two groups of seven), vaccinated with either one or two doses of the vaccine, were monitored for antibody response over one year. All sheep developed high titres of neutralizing antibodies by 35 days after first vaccination and titres were maintained over one year. Control sheep (n=7) remained seronegative until challenge. One year after vaccination, all sheep were inoculated with a virulent BTV-2. All controls developed pyrexia, clinical signs and viraemia. In contrast, the sheep vaccinated with one or two doses of inactivated BTV-2 vaccine were protected from clinical disease and viraemia was completely prevented. These data show that a single dose of the BTV-2 vaccine given to sheep induces a strong immunity which confers protection for at least one year.

Assessment of efficacy of a bivalent BTV-2 and BTV-4 inactivated vaccine by vaccination and challenge in cattle.

The efficacy of a bivalent inactivated vaccine against bluetongue virus (BTV) serotypes 2 (BTV-2) and 4 (BTV-4) was evaluated in cattle by general and local examination, serological follow-up, and challenge. Thirty-two 4-month-old calves were randomly allocated into 2 groups of 16 animals each. One group was vaccinated subcutaneously (s/c) with two injections of bivalent inactivated vaccine at a 28-day interval, and the second group was left unvaccinated and used as control. Sixty-five days after first vaccination, 8 vaccinated and 8 unvaccinated calves were s/c challenged with 1 mL of 6.2 Log10 TCID50/mL of an Italian field isolate of BTV serotype 2, while the remaining 8 vaccinated and 8 unvaccinated animals were challenged by 1 mL of 6.2 Log10 TCID50/mL of an Italian field isolate of BTV serotype 4. Three additional calves were included in the study and used as sentinels to confirm that no BTV was circulating locally. At the time of the challenge, only one vaccinated animal did not have neutralizing antibodies against BTV-4, while the remaining 15 showed titres of at least 1:10 for either BTV-2 or BTV-4. However, the BTV-2 component of the inactivated vaccine elicited a stronger immune response in terms of both the number of virus neutralization (VN) positive animals and antibody titres. After challenge, no animal showed signs of disease. Similarly, none of the vaccinated animals developed detectable viraemia while bluetongue virus serotype 2 and 4 titres were detected in the circulating blood of all unvaccinated animals, commencing on day 3 post-challenge and lasting 16 days. It is concluded that administration of the bivalent BTV-2 and BTV-4 inactivated vaccine resulted in a complete prevention of detectable viraemia in all calves when challenged with high doses of BTV-2 or BTV-4.

DNA vaccination against pseudorabies virus and bovine respiratory syncytial virus infections of young animals in the face of maternally derived immunity.

DNA vaccination represents a unique opportunity to overcome the limitations of conventional early life vaccine strategy which is restricted by the effects of maternally derived immunity. The pseudorabies virus (PRV) infection model in neonatal piglets was employed to demonstrate that a single DNA vaccination was able to prime memory humoral immune responses in the face of high concentrations of maternally derived antibodies. Immunity induced under these conditions protected against challenge with virulent PRV at the end of the fattening period, but long-term protective responses were not correlated with the kinetics of the initial serological responses. The bovine respiratory syncytial virus (BRSV) infection model in young calves was similarly studied, however the ability of DNA vaccination to prime memory humoral responses in the face of high concentrations of maternally derived antibodies was not confirmed, illustrating that the performance of DNA vaccination varies between species and/or infectious disease targets. However, in the BRSV model system it was evident that DNA vaccination could prime cell-mediated immunity in the face of high concentrations of maternally derived antibodies. Although not sufficient to ensure protection against clinical disease or viral excretion as a standalone vaccination strategy, priming by DNA vaccination was proven to establish cell-mediated immune responses for subsequent recall with an inactivated vaccine booster. Under these conditions, protection against challenge virus re-excretion was correlated with interferon (IFN) gamma-producing T-cell responses. The safety and the efficacy of DNA vaccine priming in very young animals in the face of high concentrations of maternally derived antibody provides a unique opportunity to design innovative and flexible vaccination programs to ensure uninterrupted protection under field conditions.

Point mutations in an infectious bovine viral diarrhoea virus type 2 cDNA transcript that yields an attenuated and protective viral progeny.

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.

Assessment of the clinical and virological protection provided by a commercial inactivated bovine viral diarrhoea virus genotype 1 vaccine against a BVDV genotype 2 challenge.

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.

Genetic and antigenic variability in bovine viral diarrhea virus (BVDV) isolates from Belgium.

This report describes the genetic and antigenic variability of bovine viral diarrhea virus strains isolated in Belgium. Part of the 5' untranslated region and the 5' end of the gp53 (E2) coding sequence were amplified by PCR and sequenced. Phylogenetic analysis showed that most field isolates segregated into genotypes Ib or II. Only one out of 28 field isolates belonged to genotype Ia. Interestingly, some type I strains were equally divergent from types Ia and Ib strains and clustered into additional subtypes within genotype I. Immune sera from young calves experimentally inoculated with field isolates first identified on the basis of their sequences were used in two-way neutralisation experiments. The results clearly differentiated type I from type II strains although some degree of cross-neutralisation was observed. Within type I, the new clusters could not be antigenically differentiated from the more prevalent type Ib strains or from type Ia strain NADL, suggesting that BVDV genotype I is antigenically homogeneous. The isolation of BVDV types I and II strains from cell lines and from a bovine vaccine suggest that molecular epidemiology surveillance is warranted for BVDV.

Virus neutralising antibodies against 22 bovine viral diarrhoea virus isolates in vaccinated calves.

Seven of nine colostrum deprived calves, free from bovine viral diarrhoea virus (BVDV), were vaccinated with a commercially available vaccine containing two inactivated strains of BVDV, an inactivated strain of bovine herpesvirus-1 and modified-live strains of bovine respiratory syncytial virus and para-influenza-3 virus. The two other calves were kept as controls. The virus neutralising (VN) antibodies induced by vaccination were tested against 22 antigenically diverse BVDV isolates, including reference strains and field isolates, both cytopathic and non-cytopathic, as well as genotypes I and II. The strains were isolated in Belgium, France, Germany, the United Kingdom and the USA. While there were variations in the VN titres of the individual calves against all the strains, serum from the seven animals neutralised 20 or more of the strains tested. From the results, it can be concluded that the vaccine can stimulate the production of VN antibodies capable of neutralising a wide range of European and American isolates of BVDV, including genotypes I and II.

Diversity among bovine pestiviruses.

Bovine viral diarrhoea virus (BVDV) isolates are characterized by an important genetic, antigenic and pathogenic diversity. The emergence of new hypervirulent BVDV strains in North America has provided clear evidence of pathogenic differences between BVDV strains. The origin of BVDV diversity is related to high mutation rate occurring in RNA viruses but the consequences of mutations obviously depend on the genes which are involved. Mutations in genes encoding for structural proteins of immunological importance may have practical implications. Knowledge of BVDV diversity is important for understanding the wide variety of pathogenesis of diseases caused by the virus, for monitoring the epidemiology of the different types and for the design of optimum laboratory tests and vaccines. This review focuses on the origin and consequences of BVDV diversity with regard to pathogenesis, biotypes, and antigenic and genetic variations.

Differences in experimental virulence of bovine viral diarrhoea viral strains isolated from haemorrhagic syndromes.

In the late 1980s, a new hypervirulent and epidemic form of bovine viral diarrhoea virus (BVDV) infection appeared in North America. A similar but sporadic syndrome was later reported in Europe. To compare the pathogenic characters of the North American and European hypervirulent strains, we inoculated BVDV naïve calves with BVDV strains isolated from haemorrhagic syndromes originating in Belgium, France and the USA. The experimental procedure comprised daily clinical examination and measurement of blood and virological parameters. The American BVD890/256 strain induced severe thrombocytopaenia, profuse diarrhoea and pneumonia in all calves, indicating that hypervirulent BVDV could be the primary infectious agent of pneumonia. Interestingly, a strong correlation was observed between the intense viraemia and a decreased platelet count. None of the European strains tested induced significant pathological signs, although isolated from cases presenting haemorrhagic syndrome.

Virus neutralizing antibodies against a panel of 18 BVDV isolates in calves vaccinated with Rispoval RS-BVD.

Seven of nine colostrum-deprived calves, free from infection with bovine virus diarrhoea virus (BVDV), were vaccinated with Rispoval RS-BVD on two occasions, 21 days apart, while the other two were kept as BVDV infection controls. The virus neutralizing (VN) serum antibodies induced by vaccination were tested for their ability to neutralize 18 European BVDV isolates, including laboratory reference strains and recent field isolates, both cytopathic and non-cytopathic biotypes as well as genotypes I and II. The strains were isolated in Belgium, France, Germany and the United Kingdom. While there were large variations in the vaccine-induced VN titres of the individual calves against all the strains, e.g. the titres against Osloss NCP, the European reference strain ranged from 1.7 to 6.7 (1:log2), serum from each animal was capable of neutralizing between nine and all 18 of the strains tested. Nevertheless, from the results of this study, it can be concluded that in colostrum-deprived BVDV seronegative calves, Rispoval RS-BVD can stimulate the production of VN antibodies capable of neutralizing a wide range of antigenically diverse European isolates of BVDV, including genotypes I and II.

Antigenic diversity of bovine viral diarrhoea viral isolates contradicts the concept of herd specific strain.

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.

Bovine viral diarrhoea virus induces apoptosis in blood mononuclear cells by a mechanism largely dependent on monocytes.

The induction of apoptosis by bovine viral diarrhoea virus (BVDV) was examined in bovine peripheral blood mononuclear cells (PBMC) incubated with an antigenically homologous pair of non-cytopathic and cytopathic BVDV. Our results show that the cytopathic biotype, in contrast to the non-cytopathic counterpart, induces apoptosis in PBMC. Flow cytometry analysis of cells undergoing apoptosis revealed that: (1) monocytes constitute the major cell population undergoing apoptosis; (2) cytopathic virus also induces apoptosis in BoCD4+, BoCD8+ and BoWC1+ T cells in whole PBMC cultures but not in purified T cell suspensions; (3) the degree of apoptosis of BoCD4+ and BoCD8+ T cells incubated with the cytopathic virus was significantly enhanced by the presence of monocytes. Taken together, these results suggest that bovine monocytes play an important role in apoptosis induced by cytopathic BVDV.

Persistently infected cattle stabilise bovine viral diarrhea virus leading to herd specific strains.

Animals persistently infected with BVDV are important in the epizootiology of the Bovine Viral Diarrhea (BVD) because they are a permanent source of contamination within a herd. These animals produce large quantities of virus and have, therefore, been proposed as responsible for generating antigenic variability. However, limited studies have failed to detect antigenic or genetic changes in viruses isolated at different time from persistently infected animals. One hypothesis to account for this stability is that the immunotolerance is accompanied by a selection against antigenic change. The presence of an immunotolerant persistently infected (IPI) animal in a herd would in turn lead to herd specific strains. To verify this hypothesis, we compared 17 BVDV strains isolated from IPI animals from 3 herds of Eastern Belgium. The comparison was based on the sequence of a 389 bp fragment of E2--a gene encoding for a highly variable glycoprotein. Sequences were strongly conserved within herds but were quite different between herds, indicating that BVDV herd-specific strains do exist and are associated with the presence of IPI animals.

Naturally occurring antibodies devoid of light chains.

Random association of VL and VH repertoires contributes considerably to antibody diversity. The diversity and the affinity are then increased by hypermutation in B cells located in germinal centres. Except in the case of 'heavy chain' disease, naturally occurring heavy-chain antibodies have not been described, although antigen binding has been demonstrated for separated heavy chains or cloned VH domains. Here we investigate the presence of considerable amounts of IgG-like material of M(r) 100K in the serum of the camel (Camelus dromedarius). These molecules are composed of heavy-chain dimers and are devoid of light chains, but nevertheless have an extensive antigen-binding repertoire, a finding that calls into question the role of light chains in the camel. Camel heavy-chain IgGs lack CH1, which in one IgG class might be structurally replaced by an extended hinge. Heavy-chain IgGs are a feature of all camelids. These findings open new perspectives in the engineering of antibodies.