Transplacental infection and apparently immunotolerance induced by a wild-type bluetongue virus serotype 8 natural infection.

Until recently, bluetongue (BT) virus (BTV) serotypes reportedly causing transplacental infections were all ascribed to the use of modified live virus strains. During the 2007 BT epidemic in Belgium, a significant increase in the incidence of abortions was reported. A study including 1348 foetuses, newborns and young animals with or without suspicion of BTV infection, was conducted to investigate the occurrence of natural transplacental infection caused by wild-type BTV-8 and to check the immunocompetence of newborns. BTV RNA was present in 41% and 18.5% of aborted foetuses from dams with or without suspected BTV involvement during pregnancy, respectively. The results of dam/calf pairs sampled before colostrum uptake provide evidence of almost 10% transplacental BTV infection in newborns. Apparently immunotolerant calves were found at a level of 2.4%. The current study concludes that the combined serological and real-time PCR (RT-qPCR) result of pregnant dams gives no indication of the infection status of the offspring except in the case of a double negative result. In a group of 109 calves with clinical suspicion of BT, born during the vector-free period, 11% were found to be RT-qPCR positive. The true prevalence was estimated to be 2.3%, indicating the extent of transplacental infection in a group of 733 calves of one to 4 months of age without BT suspicion. Moreover, virus isolation was successful for two newborn calves, emphasizing the need for restricting trade to BT-free regions of pregnant dams possibly infected during gestation, even if they are BTV RT-qPCR negative.

Imidazo[4,5-c]pyridines inhibit the in vitro replication of the classical swine fever virus and target the viral polymerase.

Selective inhibitors of the replication of the classical swine fever virus (CSFV) may have the potential to control the spread of the infection in an epidemic situation. We here report that 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP) is a highly potent inhibitor of the in vitro replication of CSFV. The compound resulted in a dose-dependent antiviral effect in PK(15) cells with a 50% effective concentration (EC(50)) for the inhibition of CSFV Alfort(187) (subgroup 1.1) of 1.6+/-0.4 microM and for CSFV Wingene (subgroup 2.3) 0.8+/-0.2 microM. Drug-resistant virus was selected by serial passage of the virus in increasing drug-concentration. The BPIP-resistant virus (EC(50): 24+/-4.0 microM) proved cross-resistant with VP32947 [3-[((2-dipropylamino)ethyl)thio]-5H-1,2,4-triazino[5,6-b]indole], an unrelated earlier reported selective inhibitor of pestivirus replication. BPIP-resistant CSFV carried a T259S mutation in NS5B, encoding the RNA-dependent RNA-polymerase (RdRp). This mutation is located near F224, a residue known to play a crucial role in the antiviral activity of BPIP against bovine viral diarrhoea virus (BVDV). The T259S mutation was introduced in a computational model of the BVDV RdRp. Molecular docking of BPIP in the BVDV polymerase suggests that T259S may have a negative impact on the stacking interaction between the imidazo[4,5-c]pyridine ring system of BPIP and F224.

Delivery of DNA vaccines by agarose hydrogel implants facilitates genetic immunization in cattle.

The present study demonstrates the interest of two slow-release systems as vaccination tools in cattle. Two experiments show that a first intradermal administration of one DNA vaccine dose combined with the slow-release of a second dose conduct to a priming of the bovine herpesvirus 1-specific immune response similar to the one generated by two discrete administrations 4 weeks apart. The first experiment demonstrates the efficacy of the slow-release system with well-characterized Alzet osmotic pumps, whereas the second experiment extends the same concept with innovative agarose hydrogel implants. These latter implants are cheaper and more convenient than the osmotic pumps or repeated intradermal administrations since they contribute to an efficient priming of the immune response in a single manipulation of the animals.

Detection of BVDV persistently infected animals in Belgium: evaluation of the strategy implemented.

Until now, no official bovine virus diarrhea virus (BVDV) control program has been implemented in Belgium. The only legislation dealing with the detection of BVDV-infected animals concerns the purchase of animals. A strategy of control, based on the identification and elimination of persistently infected (PI) animals and the vaccination of cows before insemination has been designed in both the Northern and the Southern part of the country. The strategy of detection of PI animals relies on PCR testing of pools of blood. Individual blood samples corresponding to the positive pools are then tested by BVDV-antigen ELISA. A first evaluation of the measures already applied in Belgium is presented. Data obtained in 2003 are presented and discussed regarding the validation of the laboratory strategy, the prevalence of positive herds, the genotype of circulating viruses, the outcome of antigen positive animals and the need for improvement of the current legislation.

Inhibition of bovine sperm-zona binding by bovine herpesvirus-1.

The purpose of the present study was to identify a potential interference of bovine herpesvirus-1 (BoHV-1) with sperm-oocyte interactions during bovine in vitro fertilization. An inhibition of almost 70% of sperm-zona binding was observed when bovine cumulus-denuded oocytes were inseminated in the presence of 10(7) 50% tissue culture infective dose/ml BoHV-1. The inhibitory effect of BoHV-1 on sperm-zona binding was mediated by an interaction of the virus with spermatozoa, but not with oocytes. Treatment of spermatozoa with BoHV-1, however, did not affect sperm motility and acrosomal status. Antiserum against BoHV-1 prevented the virus-induced inhibition of sperm-zona binding, indicating that BoHV-1 itself affects the fertilization process. In order to investigate which BoHV-1 glycoprotein(s) are responsible for the virus-sperm interaction, BoHV-1 was treated with monoclonal antibodies against the viral glycoproteins gB, gC, gD and gH prior to insemination. Anti-gC completely prevented the inhibitory effect of BoHV-1 on sperm-zona binding, while anti-gD caused a reduction of this inhibition. Further evidence for the involvement of gC and gD in the virus-sperm interaction was provided by the fact that purified gC and gD decreased sperm-zona binding in a dose-dependent way with gC being more effective than gD. These results indicated that BoHV-1 inhibits bovine sperm-zona binding by interacting with spermatozoa. The binding of BoHV-1 to a spermatozoon is mediated by the viral glycoproteins gC and gD, and therefore seems to be comparable with the mechanisms of BoHV-1 attachment to its natural host cell.

Prime-boost strategies combining DNA and inactivated vaccines confer high immunity and protection in cattle against bovine herpesvirus-1.

DNA vaccines have frequently been associated with poor efficacy in large animals. In the present study, one administration of an inactivated marker vaccine to cattle considerably boosted both humoral and cellular arms of the immune response primed with Bovine herpesvirus-1 (BoHV-1) DNA vaccines encoding glycoprotein D (gD) or gC+gD. Calves vaccinated according to the DNA prime-inactivated boost also showed significantly enhanced virological protection as compared to controls. The 4-logarithms reduction of virus shedding observed in primed-boosted animals was comparable to the one previously reported in calves immunized twice with marker vaccines. Intradermal immunization of cattle with DNA vaccines promoted a Th2-biased immune response but also primed a cellular component that was further boosted by the inactivated vaccine. Individual IgG2 titers of vaccinated calves were significantly correlated to IFN-gamma production. The immunization protocol described in the present study demonstrates the complementarity between DNA and conventional marker vaccines.

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.

Estimating the probability of freedom of classical swine fever virus of the East-Belgium wild-boar population.

A report of the Scientific Committee on Animal Health and Animal Welfare of the European Commission (CEC, 1999.) includes recommendations for setting up monitoring programmes for classical swine fever (CSF) infection in a wild-boar population, based on the assumption that one would detect at least 5% prevalence in a CSF-infected wild-boar population. This assumption, however, is not science based. We propose an alternative method to provide evidence for a wild-boar population being free of CSF and evaluate the efficiency of a surveillance programme that was implemented in Belgium in 1998. In our study, the probability of freedom of CSF-virus was estimated based on 789 samples; these were collected from wild-boars within the surveillance programme (within the three provinces which include 95% of the Belgian wild-boar population) and examined by three diagnostics methods (antibody detection, virus detection and virus RNA detection). A Bayesian framework was used for the estimation, accounting for the diagnostic test characteristics without the assumption of the presence of a gold standard. The median probability of freedom of CSF-virus was estimated at 0.970, with a 95% credibility interval of 0.149-1.000. Independent on the choice of the prior information, the posterior distributions for the probability of freedom of CSF-virus were always skewed close to the upper boundary of 1. This represents a big gain of knowledge since we did not use any prior information for the probability of freedom of CSF-virus and took the uncertainty about the accuracy of the diagnostic methods into account.

Real Time RT-PCR for the detection and quantitation of bovine respiratory syncytial virus.

A quantitative Real Time RT-PCR assay was developed to detect and quantify bovine respiratory syncytial virus (BRSV) in the respiratory tract of infected animals. A pair of primers and a TaqMan probe targeting conserved regions of the nucleoprotein gene of BRSV were designed. The detection limit of the assay was shown to be 10(3) RNA copies and standard curve demonstrated a linear range from 10(3) to 10(8) copies as well as an excellent reproducibility. The efficiency of the BRSV Real Time RT-PCR was then assessed by detecting BRSV in lungs, tracheas and bronchoalveaolar fluids (BAL) samples of experimentally infected calves. The assay was shown to be 100 times more sensitive than conventional RT-PCR and was more efficient for BRSV diagnosis. Finally, the Real Time RT-PCR was used to quantify BRSV load in BAL fluids of four experimentally infected calves for 14 days. The high sensitivity, rapidity and reproducibility of the BRSV Real Time RT-PCR make this method suitable for diagnostic and for the evaluation of the efficiency of new vaccines.

Detection of a novel border disease virus subgroup in Tunisian sheep.

Nine pestiviruses isolated from different batches of a contaminated Tunisian sheep pox vaccine and one Tunisian field ovine isolate of border disease virus (BDV) were studied at the antigenic and molecular levels. Seroneutralization tests were carried out on three vaccine isolates, the Tunisian field isolate and representative reference strains of the different pestivirus groups. The antigenic study showed that the Tunisian isolates were closer to the two BDV reference strains than to the Alfort-187 and the NADL reference strains. Comparison of the nucleotide sequences of the 5'-non coding regions of all the Tunisian isolates to those of other pestiviruses have shown that these isolates were distinct from the established pestivirus species. The entire N(pro)-E2 coding sequences of four Tunisian isolates were determined and compared to other pestiviruses. Segregation of these pestiviruses based on the N(pro)-E2 region was identical to that obtained with the 5'UTR sequences. The phylogenetic tree obtained with these sequences showed that the Tunisian isolates formed a separate branch between the BDV and CSFV groups, and consequently a possible new species within the pestivirus genus. However, as indicated by the antigenic study and the host origin of the isolates, the Tunisian isolates were assigned to a novel subgroup within the BDV species.

Effects of hypervaccination with bovine herpesvirus type 1 gE-deleted marker vaccines on the serological response and virological status of calves challenged with wild-type virus.

Twenty-four calves were immunised four times with gE-deleted infectious bovine rhinotracheitis marker vaccines before being challenged with small doses of wild-type bovine herpesvirus type 1 (BHV-1). The repeated vaccinations induced strong immunity that prevented detectable virus replication and gE-seroconversion after the challenge infection in most of the calves. The hypervaccinated calves that shed virus after the challenge infection showed no delay in gE-seroconversion compared with unvaccinated control calves. Using a sensitive nested PCR, BHV-1 gE sequences could be detected in the trigeminal ganglia of several of the gE-seronegative, challenge-infected calves, possibly indicating the presence of wild-type BHV-1 DNA.

[Clinical signs and diagnosis of a severe primary infection with BVDV subtype 1b in a dairy herd]. / Klinische verschijnselen en diagnostiek bij een ernstige primaire BVD-virus subtype 1b-infectie in een melkveekoppel.

As a result of a BVDV infection in a herd consisting of 95 adult cattle 17 cows aborted their calves within a period of 3.5 months, one third got severe diarrhoea, 3 cows died and an increased percentage of the cattle got lochiometra after calving or abortion. The disease was diagnosed by paired serological testing of cattle with diarrhoea or abortion and post mortem examination of several aborted calves. From one foetus BVDV virus was isolated and subsequently subtyped by sequencing. Of aborting cattle, the testing results were influenced by the interval between infection and abortion. These results indicate that a primary infection with BVDV subtype 1b can cause severe clinical symptoms in a dairy herd.

Real-time PCR for simultaneous detection and genotyping of bovine viral diarrhea virus.

Since two genotypes of bovine viral diarrhea viruses (BVDV) occur in Belgian herds, their differentiation is important for disease surveillance. A quantitative real-time PCR assay was developed to detect and classify bovine viral diarrhea viruses in genotype I and II. A pair of primers specific for highly conserved regions of the 5'UTR and two TaqMan probes were designed. The FAM and VIC-labeled probe sequences differed by three nucleotides, allowing the differentiation between genotype I and II. The assay detectability of genotype I and II real-time PCR assay was 1000 and 100 copies, respectively. Highly reproducible data were obtained as the coefficients of variation of threshold cycle values in inter-runs were less than 2.2%. The correct classification of genotype I and II viruses was assessed by using reference strains and characterized field isolates of both genotypes. The application to clinical diagnosis was evaluated on pooled blood samples by post run measurement of the FAM- and VIC-associated fluorescence. The 100% agreement with the conventional RT-PCR method confirmed that this new technique could be used for routine detection of persistently infected immunotolerant animals.

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.

Isolation of a glycoprotein E-deleted bovine herpesvirus type 1 strain in the field.

During a field trial to evaluate the efficacy of repeated vaccinations with bovine herpesvirus type 1 (BHV-1) marker vaccines, a glycoprotein E (gE)-negative BHV-1 strain was isolated from the nasal secretions of two cows, eight months after vaccination with a gE-negative live-attenuated vaccine, initially given intranasally, then intramuscularly. The strain isolated was characterised using immunofluorescence, restriction analysis and PCR. All the techniques used identified the isolated virus as a gE-negative BHV-1 phenotypically and genotypically identical to the Za strain used as a control.

Enhancement of the immune response and virological protection of calves against bovine herpesvirus type 1 with an inactivated gE-deleted vaccine.

Four immunisation protocols based on inactivated and attenuated commercially available marker vaccines for bovine herpesvirus type 1 (BHV-1) were compared. The first group of calves were vaccinated with an attenuated vaccine administered intranasally and an inactivated vaccine injected subcutaneously, four weeks apart; the second group were vaccinated twice with the attenuated vaccine, first intranasally and then intramuscularly; the third group were vaccinated twice subcutaneously with the inactivated vaccine; and the fourth group were vaccinated twice intramuscularly with the attenuated vaccine. A control group of calves were not vaccinated. The cellular and humoral immune responses were highest in the two groups which received at least one injection of the inactivated vaccine. Virological protection was observed in all the vaccinated groups after a challenge infection and reactivation by treatment with dexamethasone, but the calves which received one dose of the inactivated vaccine as a booster or two doses of the inactivated vaccine excreted significantly less of the challenge virus than the calves which were vaccinated only with the attenuated preparation.

Inhibition of histone deacetylases induces bovine leukemia virus expression in vitro and in vivo.

Packaging into nucleosomes results in a global transcriptional repression as a consequence of exclusion of sequence-specific factors. This inhibition can be relieved by using inhibitors of histone deacetylases, acetylation being a major characteristic of transcriptionally active chromatin. Paradoxically, the expression of only approximately 2% of the total cellular genes is modulated by histone hyperacetylation. To unravel the potential role of this transcriptional control on BLV expression, we tested the effect of two highly specific inhibitors of deacetylases, trichostatin A (TSA) and trapoxin (TPX). Our results demonstrate that treatment with TSA efficiently enhanced long terminal repeat-directed gene expression of integrated reporter constructs in heterologous D17 stable cell lines. To further examine the biological relevance of these observations made in vitro, we analyzed ex vivo-isolated peripheral blood mononuclear cells (PBMCs) from bovine leukemia virus (BLV)-infected sheep. TSA deacetylase inhibitor induced a drastic increase in viral expression at levels comparable to those induced by treatment with phorbol-12-myristate 13-acetate and ionomycin, the most efficient activators of BLV expression known to date. TSA acted directly on BLV-infected B lymphocytes to increase viral expression and does not seem to require T-cell cooperation. Inhibition of deacetylation after treatment with TSA or TPX also significantly increased viral expression in PBMCs from cattle, the natural host for BLV. Together, our results show that BLV gene expression is, like that of a very small fraction of cellular genes, also regulated by deacetylation.

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.