Comparative analysis of monoclonal antibodies against pestiviruses: report of an international workshop

Thirty-three pestivirus strains were grown in cell culture and characterized by immunostaining with 19 monoclonal antibodies (MAbs) raised against hog cholera virus (HCV), with 42 MAbs against bovine viral diarrhoea virus (BVDV) and with 13 MAbs against border disease virus (BDV). Seven MAbs reacted with all pestivirus strains tested, eight MAbs detected only the seven HCV strains, three detected only the 16 BVDV strains. No MAb was found that was specific for BDV. BVDV and BDV strains were broadly cross-reactive with the MAbs, indicating a close relationship between these two species, whereas HCV strains were characterized as distinct from BVDV and BDV.     

Epidemiological study of border disease virus infection in Southern chamois (Rupicapra pyrenaica) after an outbreak of disease in the Pyrenees (NE Spain)

In 2001 and 2002, an outbreak of a previously unreported disease, associated with a border disease virus (BDV), caused high mortality in the Southern chamois (Rupicapra pyrenaica) population in the Alt Pallars-Aran National Hunting Reserve in the Catalan Pyrenees (NE Spain). Between 2002 and 2006, sera and/or tissue samples taken from 116 healthy chamois shot during the hunting season, plus 42 from chamois affected by different diseases, were studied. A blocking enzyme-immunosorbent assay (ELISA) was used to study pestivirus seroprevalence in 114 healthy hunted and 31 diseased chamois, yielding positive results in 73.7 and 22.6% of the chamois, respectively. Comparative virus neutralization tests (VNT) performed on 42 seropositive samples with 6 pestivirus strains yielded statistically higher titres to BDV Spain 97, followed by BDV chamois, BDV 137/4, BDV Moredun, Bovine Diarrhoea virus-1 (BVDV-1) NADL and BVDV-2 atypical. Virological investigations for pestivirus detection were performed using an antigen ELISA test in 82 healthy and 18 diseased chamois, RT-PCR in 16 healthy and in all diseased chamois, and virus isolation in 14 diseased chamois. No viral antigen was detected in any of the healthy animals. A pestivirus, characterized as BDV by monoclonal antibodies, was detected in the 10 chamois showing clinical signs consistent with BDV infection. Sequence analysis in the 5' untranslated region (5'-UTR) revealed that they were grouped into the BDV-4 genotype. In the remaining chamois, infectious keratoconjunctivitis, pneumonia, trauma and contagious ecthyma were diagnosed. The cause of death was unknown in five chamois. The results suggest that the infection has become endemic in the population and that it could have a significant impact on chamois population dynamics.     

Differential diagnosis of classical swine fever and border disease: seroepidemiological investigation of a pestivirus infection on a mixed sheep and swine farm.]

During recent years neutralizing antibodies against Border Disease Virus (BDV) were found repeatedly in German pig herds. Consequently there was a demand for a differential diagnostic system. A permanent sheep cell line and BDV reference strain Moredun were chosen and were applied in a could be used case study. A pestivirus could be isolated from piglets on a mixed farm and was characterised as 'non-Classical Swine Fever' (CSF) by using monoclonal antibodies. Due to a CSF suspicion the pig herd was destroyed immediately. Serum samples of sheep from the same farm were used for further characterisation of the new virus isolate. A neutralization test of the sheep sera was performed against different pestiviruses and the new isolate. Neutralizing antibody titres against the new virus pig isolate were significantly higher than against all other pestiviruses. BDV strain Moredun recognised the antibodies clearly, whereas CSF viral strain Alfort 187 and several isolates of bovine viral diarrhoea virus (BVDV) strains scored the lowest cross reaction.     

Genetic typing of pestiviruses from New Zealand

AIM: To investigate the genetic type of 20 pestiviruses collected from New Zealand over the period 1967-97. METHODS: The pestiviruses were genetically typed by the sequencing of polymerase chain reaction (PCR) products. The primers selected were from the 5'-untranslated region (5'-UTR) of the pestivirus genome and consistently amplified a 288 bp fragment from all samples tested. RESULTS: Sequencing and phylogenetic analysis of PCR products revealed that all samples obtained from cattle represented bovine viral diarrhoea (BVDV) type I. Two sheep isolates were characterised as border disease virus (BDV). A pestivirus isolated from foetal calf serum of USA origin was typed as BVDV type II. CONCLUSIONS: The findings show that the evolution of pestiviruses in New Zealand has been similar to Europe and North America, indicating the occurrence of a conservative phylogenetic branch of BVDV type I in cattle and the presence of BDV in the sheep population.     

Ovine pestiviruses: their taxonomic status revealed by palindromic nucleotide substitutions

We examined previously identified border disease virus (BDV) strains by using a newly proposed genotyping procedure based on palindromic nucleotide substitutions (PNS) in the 5'-untranslated region (UTR), and found 22 (41.5%) out of 53 strains of BDV in the nucleotide sequence databases are not of BDV. All the 22 ovine pestivirus strains were allocated to the BVDV species according to the PNS, and were compared with reference strains of pestivirus 1 (BVDV-Ia,-Ib, and-Ic genovars), pestivirus 2 (BVDV-II genovar), pestivirus 3 (BDV) and pestivirus 4 (CSFV), respectively. Ten strains (Weybridge, A553, B1056, D771/1, D861, D1120/1, D1432/P, Q1161/1, Q1161/2, 114817) showed a palindromic structure in the 5'-UTR characteristic to the BVDV-Ia genovar, three strains (7535, 7546, 7548) were characteristic to the BVDV-Ib genovar, and nine strains (BD-78, 59386, SCP, Lees, C413, 167237, 168149, 173157, 175375) belonged to the BVDV-II genovar.     

Characterization of porcine and some ruminant pestiviruses by cross-neutralization

Serologic relationships between 11 pestivirus strains that originated from pigs and five that originated from cattle or sheep were studied by cross-neutralization. Experiments were performed with pig and sheep sera raised against the strains. The results were analysed by a computerized taxonomic procedure. The 16 viruses were classified into four distinct serologic groups. All hog cholera virus (HCV) strains were classified in one group; the other three groups consisted of strains that can infect pigs, but that are identified as bovine viral diarrhoea virus (BVDV) or border disease virus (BDV), or showed a closer relationship to BVDV and BDV than to HCV.     

Identification of bovine viral diarrhea virus type 1 in yaks (Bos poephagus grunniens) in the Himalayan region

Since cattle are widely infected by bovine viral diarrhea virus (BVDV) in India, we searched for pestivirus infection in yaks. Of 71 pure and crossbred yaks from Himalayan region, pestivirus antigen was detected by Ag-ELISA in three animals. Pestivirus in leukocyte and cell culture isolated virus samples originating from positive yaks was also confirmed by RT-PCR using panpestivirus specific primers selected from 5'-untranslated region (5' UTR). The 5' UTR, N(pro) and E2 regions were sequenced and used for genetic typing. Phylogenetic analysis revealed that pestiviruses detected in three Himalayan yaks were similar genetically, belonging to BVDV-1. Antigenic characterisation of yak pestivirus also confirmed the typing as BVDV-1. This is the first report on the identification of BVDV type 1 in yaks.     

Influence of communal alpine pasturing on the spread of pestiviruses among sheep and goats in Austria: first identification of border disease virus in Austria

The purpose of this investigation was to determine the influence of communal Alpine pasturing on the spread of pestivirus infections among sheep and goats. The study included 481 sheep from 23 farms and 131 goats from 26 farms pastured on separated Alpine meadows in the western part of Austria. At the starting of pasturing on the sheep meadow, 325 (67.6%) animals were seropositive, on the goat meadows in 16 (12.2%) samples antibodies to pestiviruses were detected. At the end of pasturing, 74 seronegative sheep and two seronegative goats had seroconverted. Between the beginning and the end of pasturing the seroprevalence in sheep increased significantly from 67.6% to 83% (P<0.05). Moreover, in the peripheral blood mononuclear cells of four sheep, pestivirus-specific RNA was detected before as well as after pasturing; these animals remained serologically negative throughout the investigation. They were, therefore, identified as persistently infected. Sequence analysis in the N(pro) region revealed that the detected pestiviruses were the same at genetic level and they were grouped into the Border disease virus (BDV)-3 genotype. No pestivirus RNA was found in goat samples. The results of this survey indicate that communal Alpine pasturing does play a key role in the spread of BDV. Moreover, BDV has been identified and characterized for the first time in sheep in Austria, which until then had been regarded as being free from BD.     

Flock-prevalence of border disease virus infection in Basque dairy-sheep estimated by bulk-tank milk analysis

Bulk-tank milk (BTM) samples from 154 sheep flocks were used to estimate BDV prevalence in the Basque Country in Spain using an ELISA and a RT-PCR test. The proportion of antibody-positive flocks was 68% but varied significantly between provinces and was 93% in Araba and 54-55% in Bizkaia and Gipuzkoa. Most ELISA-positive flocks had very low antibody inhibition percentage (AIP) indicating high seroprevalence and recent BDV exposure. However, only 9% flocks were PCR-positive suggesting few infected ewes were being milked at the time of sampling. Phylogenetic analysis of the 5' NCR sequences of BDV from seven infected flocks showed that all except one clustered within the group formed by BDV type C strains from a previous study in the region, whereas the remaining isolate was closest to BDV type A. These results suggest that BDV strains in most Basque flocks have a common origin and differences in prevalence between provinces are associated to recent events affecting BDV spread such as use of communal pastures and sheep trading. The widespread distribution of BDV in the region, advocates for the implementation of BDV control strategies and highlights the potential risk of sheep as a pestivirus reservoir for other species.     

Retrospective study of pestivirus infection in Pyrenean chamois (Rupicapra pyrenaica) and other ungulates in the Pyrenees (NE Spain)

In 2001 a new Pestivirus (Family Flaviviridae) was associated with an outbreak of a previously unreported disease in Pyrenean chamois (Rupicapra pyrenaica) in the Pyrenees (NE Spain). Molecular characterization assigned this virus to the Border Disease Virus (BDV) cluster, BDV-4 genotype. A retrospective study was performed in archived sera and spleen of 74 Pyrenean chamois and in archived sera of 28 mouflon (Ovis ammon), 56 red deer (Cervus elaphus), 43 roe deer (Capreolus capreolus) and 29 fallow deer (Dama dama) from the Pyrenees between the years 1990 and 2000. Thirty six of 74 (48.6%) sera of Pyrenean chamois, one of mouflon and one of red deer were positive by an ELISA antibody test. Comparative virus neutralization tests were performed on 26 seropositive chamois, one mouflon and one red deer, using five pestivirus strains. An ELISA antigen test was performed on 37 seronegative chamois and yielded positive results in one chamois and inconclusive result in two. RT-PCR and virus isolation performed on spleen samples from these three animals gave positive results in the positive and one inconclusive animal. Sequence analysis in the 5' unstranslated region revealed that they were grouped into the BDV-4 genotype. Virological and serological data of the present study indicate that BDV infection has been present in the chamois population since at least 1990, 11 years before the first outbreak of disease. Therefore, the emergence of the disease in 2001 is apparently due to other factors rather than the introduction of a new virus in the chamois population.     

Antigenic variation and genotype of isolates of porcine reproductive and respiratory syndrome virus in Korea

A panel of three anti-glycoprotein 5 (gp5) protein monoclonal antibodies (mAbs) (15, 28 and 246) and three anti-nucleocapsid (N) protein mAbs (SDOW17, VO17 and EP147) was used to investigate, by an indirect fluorescent antibody test, the antigenic variations of 50 Korean isolates of porcine reproductive and respiratory syndrome virus (PRRSV), and compare them with a us ATCC vR2332-derived attenuated vaccine strain and the reference European Lelystad strain of PRRSV. A multiplex PCR assay for the differentiation of European and North American genotypes of PRRSV was used to determine the genotype of the 50 Korean isolates. Forty-six (92 per cent) of the 50 Korean isolates shared the epitopes recognised by the anti-N protein mAb SDOW17. No reactivity to the anti-gp5 and anti-N protein mAbs was observed with the other four isolates. Six distinct patterns could be identified on the basis of their reactivities with the anti-PRRSV mAbs. All 50 isolates were identified as North American genotypes by the differential PCR.     

Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies

Nineteen monoclonal antibodies (MAbs) with specificity for hog cholera virus (HCV) were prepared. They were used in an immune binding (peroxidase linked) assay to determine the reaction patterns of HCV isolates from Europe, Brazil, USA, Japan and Malaysia, as well as laboratory reference strains of the virus. A further panel of 17 MAbs raised against bovine virus diarrhoea virus (BVDV) was included in the study, together with 5 MAbs raised against a non-HCV pestivirus of porcine origin. All the MAbs were also tested against representative strains of BVDV and border disease virus. Six MAbs were HCV-specific, reacting with all isolates of HCV and none of the ruminant viruses. Among the other HCV MAbs geographical variation in reaction patterns was observed. There was evidence of antigenic distinction between recent European isolates, and archive material originally isolated more than 10 years ago.     

Identification and molecular characterization of border disease virus (BDV) from sheep in India

Pestiviruses isolated from sheep and goats in India thus far have been bovine viral diarrhoea virus 1 (BVDV-1) or BVDV-2. During routine genetic typing of pestiviruses in the years 2009-10, border disease virus (BDV) was detected in eight Indian sheep of a flock showing clinical signs of BD by real time RT-PCR. All the samples yielded positive virus isolates in cell culture but were found negative by a BVDV antigen ELISA. A representative BDV isolate was characterized at genetic and antigenic level. Phylogenetic analysis carried out in 5′-UTR, N^pro and E2 regions of genome typed the Indian BDV isolate as BDV-3. A more detailed analysis in N^pro and entire region coding structural proteins showed that the N^pro (168), C (100 aa), E^rns (227 aa), E1 (195 aa) and E2 (373 aa) proteins were of size characteristic for BDV reference strain X818. Antigenic differences were evident between the BDV-3 isolate and previously reported BDV-1, BDV-5 and BDV-7 strains. Although origin of BDV-3 in India is not clear, the results reflect probable introduction through trade in sheep between India and other countries or BDV-3 may be more widely distributed. Additionally, this study suggests that for diagnosis of BDV infection, the commercial BVDV Ag-ELISA should be used with caution. This is the first identification of BDV in sheep in India which highlights the need for continued pestivirus surveillance and assessing its impact on sheep and goat production.     

Seroprevalence of antibodies to ruminant pestiviruses in sheep and goats in Tyrol (Austria)

In this study 2058 blood samples from sheep of 150 flocks from the province of Tyrol were tested by ELISA and serum neutralisation tests for antibodies to ruminant pestiviruses. In the ELISA, positive results were obtained with 34.9% of individual sheep sera and in 89.3% of the sheep flocks. The prevalence in sheep and sheep flocks varied according to areas. Seroprevalence of pestiviruses was significantly (p < 0.05) higher in small ruminants pastured during summertime on the Alps. Comparative neutralisation studies were carried out on all positive blood samples with BVDV-1, BVDV-2 and BDV. 443 seropositive sheep samples exhibited clearly the highest titre against one of the pestivirus strains tested. 413 revealed the highest titres (2 or more fold) to BVDV-1, 6 to BVDV-2 and 24 to BDV. In some areas a very high rate of pestivirus seroprevalence could be found. This fact could be harmful to the BVDV-Elimination and Controlling Program in cattle in Austria.     

Bovine viral diarrhoea virus infections in piglets born to sows vaccinated against swine fever with contaminated vaccine

On eight farms a congenital pestivirus infection in piglets was detected which could be traced to vaccination of the dams against swine fever (SF). Viruses isolated from the piglets were not recognised by monoclonal antibodies (MCAs) against swine fever virus (SFV) and were shown to be bovine viral diarrhoea virus (BVDV) or Border disease virus. The expected 'Chinese' strain of SFV could not be demonstrated in the batch of vaccine that had been used on these farms. Instead, a contaminating pestivirus was recovered which was not recognised by the MCAs against SFV. The contaminant had an unexpectedly high virulence for lambs and induced antibodies against BVDV in lambs and pigs. It could, therefore, be characterised as BVDV or Border disease virus.     

Serological survey for antibodies against pestiviruses in sheep in Austria

The prevalence of antibodies to pestiviruses was investigated in 4931 sheep, in 377 flocks, in four federal states of Austria, by means of an indirect elisa that detected antibodies to Border disease virus (BDV) and bovine viral diarrhoea virus (BVDV). The mean flock prevalence was 62.9 per cent and the mean individual prevalence was 29.4 per cent. Comparative neutralisation studies on the elisa-positive samples with BVDV type 1 (BVDV-1), BVDV type 2 (BVDV-2) and BDV recorded 336 samples with higher titres (more than four times average) to BVDV-1, three samples with higher titres to BVDV-2 and 55 samples with higher titres to BDV. The other samples did not show clear differences in antibody titres against the strains of pestivirus tested because of cross-reactions. The seroprevalence of pestiviruses in sheep was significantly higher on farms with cattle. There were significant regional differences between the prevalences in flocks and individual sheep, the highest prevalences being in the region of Austria where communal alpine pasturing of sheep, goats and cattle is an important part of farming.     

Porcine reproductive and respiratory syndrome virus (PRRSV)-specific mAbs: supporting diagnostics and providing new insights into the antigenic properties of the virus

The porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important viral pathogens in the swine industry. Despite great efforts of pig holders, veterinarians, researchers and vaccine developers, the virus still causes major production losses. It is clear that efficient and correct monitoring and rational development of vaccines are crucial in the combat against this pathogen. PRRSV-specific monoclonal antibodies (mAbs) are essential tools for both diagnostic and research purposes. This study describes the production of PRRSV GP3-, GP5- and N-specific hybridomas and an extensive characterization of the mAbs. The N-specific mAbs generated in this study appear to be useful tools for diagnostics, as they were found to react with genetically very different PRRSV isolates and may serve to discriminate between European and American type PRRSV isolates. These mAbs also allowed detection of the PRRSV N protein in both formalin-fixed, paraffin-embedded tissue sections and frozen tissue sections of PRRSV-infected lungs, further illustrating their diagnostic value. Different neutralization assays pointed out that none of the GP3- and GP5-specific mAbs tested shows virus-neutralizing capacity. This is noteworthy, as these mAbs recognize epitopes in the predicted ectodomains of their target protein and since the GP5-specific antibodies specifically react with the antigenic region that corresponds to the "major neutralizing epitope" suggested for American type PRRSV. The current findings argue against an important role of the identified antigenic regions in direct antibody-mediated neutralization of European type PRRSV in vivo. However, it is also clear that findings concerning a specific PRRSV epitope cannot always be generalized, as the antigenic determinants and their biological properties may differ radically between different virus isolates.     

Prevalence of antibodies to pestiviruses in goats in Austria

Serological investigations were carried out to determine the prevalence of pestiviral infections in goats in Austria, and to investigate the possible relations to herd management practices. The prevalence of antibodies to pestiviruses was investigated in 549 goats in 80 flocks from four regions of Austria. The examination for antibodies was performed using an indirect enzyme-linked immunosorbent assay detecting antibodies to the border disease virus (BDV) and bovine viral diarrhoea virus (BVDV). The observed individual prevalence was 11.5% and the flock prevalence was 31.3%. Comparative neutralization studies on the 63 seropositive samples with BVDV-1, BVDV-2 and the BDV yielded in 32 samples higher titres (> or =4-fold) to BVDV-1 and in two samples to BDV. The remaining samples did not show distinct differences in antibody titres against the pestivirus strains tested because of the cross-reactions. There was a significant (P < 0.05) association between the prevalence of antibodies to pestiviruses and the presence of cattle on the farm. Significant (P < 0.05) geographical variations in individual prevalence were obtained, ranging from 3.5% in lower Austria to 20.2% in Vorarlberg.