Recombinant Newcastle disease viruses with targets for PCR diagnostics for rinderpest and peste des petits ruminants.

Since February 1st 2011, rinderpest (RP) has been officially declared eradicated worldwide. National authorities have been requested to destroy all their RP related materials. Nonetheless, their national reference laboratories performing real time reverse transcription polymerase chain reaction assays (PCR diagnostics) need RP positive control samples, since some countries still prefer to maintain diagnostic capability for RP for several reasons. In the future, a similar situation will arise for peste des petits ruminants (PPR) as the ambition has been expressed to eradicate PPR. Anticipating on this, we intended to perform qualified PCR diagnostics without use of infectious RPV or PPRV. Therefore, Newcastle disease virus (NDV) with small RNA inserts based on RPV or PPRV sequences were generated and used as positive control material. Recombinant NDVs (recNDVs) were differentially detected by previously established PCR diagnostics for RPV or PPRV. Both recNDVs contain a second PCR target showing that additional targets in NDV are feasible and would increase the diagnostic sensitivity by use of two PCR assays. RecNDV with small PCR targets is not classified as RPV or PPRV containing material, and can be used to mimic RPV or PPRV. Using these recNDVs as virus positive material contributes to the ambition of worldwide eradication, while qualified PCR diagnostics for these OIE-listed diseases remains operational.

Bluetongue virus with mutated genome segment 10 to differentiate infected from vaccinated animals: a genetic DIVA approach.

Bluetongue virus (BTV) includes 24 serotypes and recently even more serotypes are proposed. Mass vaccination campaigns highlight the need for differential diagnostics in vaccinated populations. Bluetongue disease is routinely diagnosed by serological and virological tests by which differentiation infected from vaccinated animals (DIVA principle) is not possible. Real time PCR tests preferably detect all BTV serotypes (panBTV PCR tests). These PCR tests operate as frontline test to detect new BTV incursions. However, highly sensitive panBTV PCR tests can also detect currently applied inactivated and modified-live vaccines. Here, BTV with eight silent mutations in segment 10 (Seg-10) was generated by reverse genetics. This BTV mutant is not detected by a Seg-10 panBTV PCR test (genetic DIVA). Thus, inactivated BT vaccine with this mutated Seg-10 will avoid false positive PCR results post vaccination, whereas BTV infected animals can be positively diagnosed with the accompanying Seg-10 panBTV PCR test (DIVA-test) far beyond the infectious period.

Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of Bovine viral diarrhea virus type 1 and 2.

Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent characterization of both strains revealed major antigenic differences. Because the envelope glycoprotein E2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by constructing chimeric BVDV type 1 and 2 E2 genes in expression plasmids. These plasmids with chimeric E2-genes were transfected in SK6 cells and transient expression was studied by immunostaining with a panel of MAbs specific for E2 of BVDV1 or BVDV2, resulting in the localization of type-specific antigenic domains at similar regions. These results indicate that E2 glycoproteins of both BVDV types exhibit a comparable antigenic structure, but with type specific epitopes. In addition, the antigenic resemblance with envelope glycoprotein E2 of Classical swine fever virus is discussed.

Bluetongue virus serotype 8 (BTV-8) infection reduces fertility of Dutch dairy cattle and is vertically transmitted to offspring.

In 2007, BTV-8 re-emerged for the second year in the Netherlands and caused morbidity and increased mortality in cattle herds. In addition, cattle farmers reported reduced fertility in their cows. For this study, fifteen herds that were not vaccinated were selected. These were matched to 10 vaccinated herds by geographic region. At the start of the study, in July 2008, all cattle in the non-vaccinated herds >1 year old were sampled. All seronegative cows entered the study program and blood samples from these cows were tested for antibodies against BTV-8 in an ELISA. Cows were sampled at intervals of three weeks and sampling was stopped once a cow tested seropositive. Sampling ceased in all remaining cows in December 2008. Newborn calves originating from infected dams or from vaccinated dams were tested by PCR for BTV-8. Fertility data were obtained from the Royal Dutch Cattle Syndicate (CRV). Multi-level generalized latent and linear models were used for analyses. In 2008, 185 (17.2%) out of 1,074 initially seronegative non-vaccinated cattle seroconverted and were assumed to be infected with BTV-8. Infected cows were 5 (95% CI: 1.9-14.3) times more likely to return for insemination within 56 days after first insemination. In addition, these cows needed 1.7 (95% CI: 1.4-2.0) times more inseminations for an assumed pregnancy, and needed 2.5 (95% CI: 2.4-2.6) times more days between first and last insemination compared to the period prior to seroconversion and compared to cows not infected by BTV-8 in 2008. No association between BTV-8 infection and the chance to abort between 100 and 260 days after last insemination was found. In total, 48 calves originating from infected cows were tested by PCR for the presence of BTV-8. Ten (20.8%) out of these 48 calves were born PCR-positive. None of 256 calves from vaccinated dams tested PCR-positive. Further, cows infected during the second half of gestation had a 15.5 times (95% CI: 1.3-190.4) higher chance of a PCR-positive newborn calf compared to cows infected in the first half of gestation. This study showed that BTV-8 has a negative effect on fertility of dairy cattle.

Evaluation of an indirect ELISA for detection of antibodies in bulk milk against bluetongue virus infections in the Netherlands.

After the introduction of bluetongue virus serotype 8 (BTV-8) in western Europe in 2006, an indirect ELISA for detection of serogroup-specific antibodies against BTV in serum samples was validated for individual milk samples by the Central Veterinary Institute and the Animal Health Service in the Netherlands (Kramps et al., 2008). In order to develop a cost-effective monitoring tool, we now have evaluated this ELISA also for use in bulk milk. Therefore, bulk milk samples and individual milk samples were collected from 92 herds in the affected southern region in the Netherlands in 2007, before the start of the vaccination campaign. In addition, bulk milk samples collected from 88 herds before the bluetongue introduction in 2006 ("historically negative" samples) have been tested. With these results ROC analyses were performed and herd specificity and herd sensitivity of the bulk milk ELISA were estimated. All "historically negative" bulk milk samples were negative in the ELISA, with a mean S/P ratio of 10 ± 0.8%. The herd sensitivity and herd specificity of the ELISA in bulk milk samples depend on the cut-off that is chosen. In order to detect a within-herd-prevalence of 1%, the optimal cut-off S/P ratio 13% was found. A few herds with one or two milk-positive animals would then be missed. The specificity will be 100%. A within-herd-prevalence of 10% can be detected with 100% sensitivity at a cut-off S/P ratio of 96%. In conclusion, the indirect ELISA in bulk milk samples is a very specific and sensitive test which can be implemented in monitoring and surveillance systems in unvaccinated populations.

Questionnaire survey about the motives of commercial livestock farmers and hobby holders to vaccinate their animals against Bluetongue virus serotype 8 in 2008-2009 in the Netherlands.

After a massive epidemic of Bluetongue virus serotype 8 (BTV-8) among ruminants in 2006-2007 in the European Union (EU), the Netherlands started a voluntary emergency vaccination campaign in May 2008, subsidized by the EU. At the start of a new campaign in 2009, without subsidized vaccination, we investigated by mail survey the motives of farmers and hobby holders to vaccinate against BTV-8 in 2008 and 2009. Mean vaccine uptake in 2008 was: 73% in sheep, 71% in cattle, 43% in goat farms and 67% in hobby holdings. Top-5 motives pro-vaccination were: prevention of production loss; subsidized vaccination; recommendation by practitioner; welfare reasons; contribution to the eradication campaign. Top-5 motives against vaccination were: vaccination costs; absence of clinical BT-problems; presumed low infection risk; balance between vaccination costs and loss without vaccination; bad experience with earlier vaccination campaigns. Willingness to vaccinate was significantly lower in 2009: 42% in sheep, 58% in cattle, 19% in goat farms and 49% in hobby holdings. Measures to stimulate vaccination among those that did not want to vaccinate in 2009 were: subsidized vaccination; possibility to vaccinate their own animals; more information on efficacy/safety of vaccine and why animals had to be vaccinated again; availability of a BT vaccine combined with vaccine(s) against other diseases.

Vertical transmission of bluetongue virus serotype 8 virus in Dutch dairy herds in 2007.

In February 2008, evidence was found for transplacental infection of bluetongue virus serotype 8 (BTV-8) in PCR negative, seropositive heifers in Northern Ireland originating from the Netherlands. The relevance of this route of transmission was studied in Dutch cow-calf combinations in the Netherlands of which the calves were born in the same time period of the year as the calves from the exported heifers, the first quarter of 2008. Blood samples were tested from 385 cows and their calves, housed in 43 dairy farms that became naturally infected with BTV-8 for the first time in 2007. All calves were at least 10 days old at the moment of first testing. In total 229 cows tested seropositive for BTV-8. Eight of these cows were still PCR positive. Out of the 229 seropositive cows, 37 calves (16.2%; 95% CI: 11.4-21.0) were tested PCR positive in the first sample taken in April 2008. In the first week of June, 34 out of the 37 PCR positive calves were still available for resampling. Three calves were still PCR positive; one was 5 months old, the other two were 3 months old. One month later, in the first week of July, all initially PCR positive calves, including the three still tested positive 1 month earlier, were PCR negative. We showed that BTV-8 can be vertically transmitted from cow to calf and can result in healthy looking viraemic calves remaining PCR positive for up to 5 months. These PCR positive calves could play a role in the epidemiology, and in particular in overwintering of BT. However, further investigations are needed to evaluate the importance of this route of transmission.

Performance of clinical signs to detect bluetongue virus serotype 8 outbreaks in cattle and sheep during the 2006-epidemic in The Netherlands.

The performance of clinical signs as a diagnostic test for the detection of BTV-8 outbreaks during the 2006-epidemic in The Netherlands was evaluated by constructing and analysing receiver operating characteristic (ROC) curves. The area under the ROC curve of the BT-associated clinical signs in cattle was 0.77. An optimal efficient test (maximising both sensitivity and specificity) in cattle herds combined a sensitivity (Se) of 67% with a specificity (Sp) of 72%, comprising the following clinical signs: ulcerations and/or erosions of oral mucosa or erosions of lips/crusts in or around nostrils or oedema of the nose or hyperaemic/purple coloration of tongue, tongue protrusion or coronitis or apathy/tiredness or muscle necrosis, stiffness of limbs or loathing or refusal to move, prostration or torticollis or anoestrus. The area under the ROC curve of the BT-associated clinical signs in sheep was 0.81. The optimal efficient test in sheep flocks combined a Se of 76% with a Sp of 72%, comprising the following clinical signs: ulcerations of oral mucosa or serous nasal discharge or erosions/ulceration of tongue mucosa or hypersensitivity of the skin or muscle necrosis, stiffness of limbs or coronitis or grinding of teeth or salivation or weakness/paresis.

A common neutralizing epitope on envelope glycoprotein E2 of different pestiviruses: implications for improvement of vaccines and diagnostics for classical swine fever (CSF)?

The Pestivirus genus within the family of Flaviviridae consists of at least three species; classical swine fever virus (CSFV) found in swine and wild boar, bovine viral diarrhoea virus type 1 and type 2 (BVDV-I and BVDV-II) mainly isolated from cattle and border disease virus (BDV) preferably replicating in ovine species. Many features demonstrate differences between CSFV and other pestiviruses, BVDV-I, BVDV-II and BDV, here defined as nonCSFV, whereas other features show similarities between all different species of pestiviruses. Focussing on the major envelope glycoprotein E2, the immunodominant protein of pestiviruses, CSFV seems to be a more distinct species within the Pestivirus genus. Here we confirm on one hand the more separated grouping of CSFV by isolation of monoclonal antibodies (MAbs) raised against E2 of BVDV-I and BVDV-II. None of these MAbs recognize E2 of CSFV strains. On the other hand, only one MAb, MAb 912, was isolated against E2 of BDV. MAb 912 binds to E2 of CSFV strains and partly neutralizes CSFV. The epitope of MAb 912 is mapped in antigenic domain B of CSFV-E2. This common epitope of CSFV strains and nonCSFV strains could have implications for development of DIVA vaccines and serological diagnostics for CSF.

Significance of the oligosaccharides of the porcine reproductive and respiratory syndrome virus glycoproteins GP2a and GP5 for infectious virus production.

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains four glycoproteins, GP(2a), GP(3), GP(4) and GP(5), the functions of which are still largely unresolved. In this study, the significance of the N-glycosylation of the GP(2a) and GP(5) proteins of PRRSV strain LV was investigated. Both glycoproteins contain two predicted N-glycosylation sites that are highly conserved between North American-type and European-type PRRSV. Using site-directed mutagenesis, single and double mutant full-length PRRSV cDNA clones were generated. After analysing the expression of the mutant proteins and the actual use of the four putative glycosylation sites in the wild-type proteins, the production of mutant virus particles and their infectivities were investigated. The results showed that the N-linked glycans normally present on the GP(2a) protein are not essential for particle formation, as is the oligosaccharide attached to N53 of the GP(5) protein. In contrast, the oligosaccharide linked to N46 of the GP(5) protein is strongly required for virus particle production. The specific infectivities of the mutant viruses were investigated by comparing their infectivity-per-particle ratios with that of wild-type virus. The results showed that the lack of either one or both of the N-linked oligosaccharides on GP(2a) or of the oligosaccharide attached to N53 of GP(5) did not significantly affect the infectivities of the viruses. In contrast, the two recombinant viruses lacking the oligosaccharide bound to N46 exhibited a significantly reduced specific infectivity compared with the wild-type virus. The implications of the differential requirements of the modifications of GP(2a) and GP(5) for PRRSV assembly and infectivity are discussed.

Oral transmission of porcine reproductive and respiratory syndrome virus by muscle of experimentally infected pigs.

The current study was performed to determine if porcine reproductive and respiratory syndrome virus (PRRSV) could be transmitted to pigs by feeding muscle tissue obtained from recently infected pigs. Muscle obtained from pigs infected with either a European strain (EU donor pigs) or American strain (US donor pigs) of PRRSV was fed to PRRSV-free receiver pigs. The donor pigs were slaughtered 11 days post-infection (dpi). PRRSV was detected by conventional virus isolation in muscle at 11 dpi from 7 of 12 EU donor pigs and 5 of 12 US donor pigs. In contrast to conventional virus isolation, all muscle samples from infected pigs were positive for viral nucleic acid by PCR, except for muscle from one animal infected with the American strain of PRRSV. Five hundred grams of raw semimembranosus muscle from each of the donor pigs was fed over a 2 days period (250 g per day) to each of two receiver pigs (48 receiver pigs). The receiver pigs were housed separately in five groups. One of the five groups was fed muscle obtained from US donor pigs that was also spiked with the American strain of PRRSV. Sentinel pigs were placed in-contact with the group of receiver pigs fed spiked muscle. All receiver pigs became viraemic by 6 days post-feeding (dpf). There was evidence of horizontal transmission with sentinel pigs, in-contact with receiver pigs, becoming viraemic. The study demonstrates that PRRSV could be infectious through the oral route via the feeding of meat obtained from recently infected pigs.

The major envelope protein, GP5, of a European porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its N-terminal ectodomain.

A set of neutralizing monoclonal antibodies (mAbs) directed against the GP(5) protein of European type porcine reproductive and respiratory syndrome virus (PRRSV) has been produced previously (Weiland et al., 1999). This set reacted with a plaque-purified virus (PPV) subpopulation of Dutch isolate Intervet-10 (I-10), but not with the European prototype PRRSV LV. In order to map the neutralization epitope in the GP(5) protein of the PPV strain, the ORF5 nucleotide sequence of PPV was determined. When the amino acid sequence derived from this nucleotide sequence was compared with that of PRRSV LV, four amino acid differences were found. Using site-directed mutagenesis, we showed that a proline residue at position 24 of the GP(5) sequence of the PPV strain enabled recognition by the neutralizing mAbs. Pepscan analysis demonstrated that the epitope recognized by the neutralizing mAbs stretched from residues 29 to 35. Surprisingly, the reactivity of the mAbs in the Pepscan system was independent of the presence of a proline in position 24. Moreover, residue 24 is located within the predicted signal peptide, implying that either the signal peptide is not cleaved or is cleaved due to the presence of Pro(24) such that the epitope remains intact. Our results demonstrate the presence of a neutralization epitope in the N-terminal ectodomain of the GP(5) protein of PRRSV and imply a role for the ectodomain of GP(5) in the infection of PRRSV.

Safety and protective efficacy of porcine reproductive and respiratory syndrome recombinant virus vaccines in young pigs.

Three porcine reproductive and respiratory syndrome virus (PRRSV) recombinants, generated by mutagenesis of an infectious cDNA clone of the Lelystad virus (LV) isolate, were tested for their safety and protective efficacy as potential PRRSV vaccines in pigs. Recombinant vABV688 contains two amino acid substitutions in the minor structural protein GP(2) resulting in improved growth on cell line CL2621; in recombinant vABV707 the region encoding the ectodomain of the major unglycosylated membrane protein M has been replaced by that of the murine lactate dehydrogenase-elevating arterivirus; recombinant vABV746 lacks the six C-terminal amino acids of the nucleocapsid protein N. First, we determined the safety of these recombinant viruses by monitoring the stability of the introduced mutations in 8-week-old pigs. We showed that the introduced genomic mutations were maintained throughout the viraemic period. Second, the protective efficacy of immunization with the recombinant viruses against challenge with a homologous and a heterologous PRRSV strain was determined in two pigs and compared with the efficacy of vABV437, a virus derived from the parental LV cDNA. The viraemia in pigs immunized with the recombinant viruses was reduced compared to pigs immunized with vABV437. In addition, the length of viraemia was reduced in the sentinel pigs that were introduced into the groups immunized with vABV746, vABV688, and vABV707, however, all of the sentinel pigs became infected. Pigs immunized with vABV707 and vABV437 were protected against challenge with homologous virus LV-Ter Huurne and transmission of the latter virus. None of the immunized pigs were protected against heterologous challenge with the virulent US isolate SDSU#73, but the vABV707- and vABV746-immunized pigs were protected against transmission of this virus from challenged pigs. In conclusion, the obtained viral recombinants are interesting candidates to be further explored for their use as vaccines against PRRSV.

Identification of porcine alveolar macrophage glycoproteins involved in infection of porcine respiratory and reproductive syndrome virus.

The aim of this study was to identify the receptor(s) for PRRSV on porcine alveolar macrophages (PAMs) by producing monoclonal antibodies (MAbs) against these cells. Hybridoma supernatants were selected for their ability to block PRRSV infection. Four MAbs, 1-8D2, 9.4C7, 9.9F2, and 3-3H2 inhibited infection and recognised cell surface, PAM-specific antigens as shown by immunofluorescence and immunoperoxidase monolayer assay. These MAbs were then used to identify cellular proteins involved in PRRSV infection by radioimmunoprecipitation assays (RIPAs). MAbs 1-8D2 and 9.9F2 each recognised a 150 kDa-polypeptide doublet, while MAbs 9.4C7 and 3-3H2 both recognised a 220 kDa-polypeptide. Glycosidase treatment demonstrated all these polypeptides to be N-glycosylated. Thus, multiple glycoproteins appear to be involved in infection of PAMs by PRRSV.

Experimental non-transmissible marker vaccines for classical swine fever (CSF) by trans-complementation of E(rns) or E2 of CSFV.

Three mutants with deletions in the E2 gene of the infectious DNA copy of the classical swine fever virus (CSFV) strain-C were constructed: one missing the B/C domain of CSFV-E2 between amino acids (aa) 693 and 746, one missing the A domain between aa 800 and 864, and one missing the complete E2 between aa 689 and 1062. All three CSFV-E2 deletion mutants were unable to generate viable virus, indicating that each of the antigenic domains of E2 is essential for viability of CSFV. To rescue the CSFV-E2 deletion mutants SK6 cell lines constitutively expressing glycoprotein E2 of CSFV were generated. The rescued viruses infected and replicated in SK6 cells as demonstrated by expression of viral proteins, but this primary infection did not result in reproduction of infectious virus. Thus, these E2 complemented viruses are considered non-transmissible. In previous experiments, we showed that simultaneous injection of E(rns) complemented virus (Flc23) via intradermal (ID), intramuscular (IM) or intranasal (IN) routes conferred protection to pigs against a lethal challenge with CSFV [J. Virol. 74 (2000) 2973]. Here, we evaluate different routes of application (ID, IM or IN) with E(rns) complemented virus Flc23 in order to find the best route for complemented CSFVs. Intradermal injection with Flc23 protected pigs against a lethal CSFV challenge, whereas intramuscular injection induced partial protection, and intranasal injection did not mediate a protective immune response in pigs at all. We used the intradermal route of vaccination to test the E2 complemented viruses. Vaccination of pigs via the intradermal route with the E2 complemented CSFVs also resulted in the induction of antibodies and in (partial) protection against CSFV challenge. Pigs vaccinated with E2 complemented virus Flc4 (deletion B/C domain) survived a lethal CSFV challenge, whereas partial protection was induced in pigs vaccinated with Flc47 (deletion E2) or Flc48 (deletion A domain) E2 complemented viruses. Serological data demonstrate that these E2 complemented mutant viruses are, in combination with well known diagnostic tests based on E2, potential marker vaccines for CSF.

Chimeric classical swine fever viruses containing envelope protein E(RNS) or E2 of bovine viral diarrhoea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response.

Three chimeric classical swine fever virus (CSFV)/bovine viral diarrhoea virus (BVDV) full-length DNA copies were constructed, based on the infectious DNA copy of the CSFV vaccine strain C. The antigenic region of E2 and/or the complete E(RNS) gene were replaced by the analogous sequence of BVDV II strain 5250. Viable chimeric virus Flc11, in which E(RNS) was replaced, was directly recovered from supernatant of SK6.T7 cells transfected with full-length DNA. Viable chimeric virus Flc9, in which E2 was replaced, resulted in recovery of virus only when SK6.T7 transfected cells were maintained for several passages. However, no virus could be recovered after replacement of both E(RNS) and E2, even after 10 cell passages. Both Flc9 and Flc11 grow in swine kidney cells (SK6), stably maintain their heterologous BVDV sequences and, as assessed by monoclonal antibody typing and radio-immunoprecipitation assays, express their heterologous proteins. Flc9 showed a slower growth rate on SK6 cells than Flc11 and wild-type Flc2 virus. Replacement of E(RNS) or E2 of C-strain-based chimeric viruses did not alter cell tropism compared to wild-type C-strain virus for SK6 and FBE cells. Both Flc9 and Flc11 induced E2 or E(RNS) antibodies, which could be discriminated from those induced after wild-type virus infection, even after repeated vaccination. Furthermore, pigs were completely protected against a lethal CSFV challenge. These results indicate the feasibility of introduction of marker antigens in a live-attenuated marker C-strain vaccine for CSFV.