In mid September 2008, clinical signs of bluetongue (particularly coronitis) were observed in cows on three different farms in eastern Netherlands (Luttenberg, Heeten, and Barchem), two of which had been vaccinated with an inactivated BTV-8 vaccine (during May-June 2008). Bluetongue virus (BTV) infection was also detected on a fourth farm (Oldenzaal) in the same area while testing for export. BTV RNA was subsequently identified by real time RT-PCR targeting genome-segment (Seg-) 10, in blood samples from each farm. The virus was isolated from the Heeten sample (IAH “dsRNA virus reference collection” [dsRNA-VRC] isolate number NET2008/05) and typed as BTV-6 by RT-PCR targeting Seg-2. Sequencing confirmed the virus type, showing an identical Seg-2 sequence to that of the South African BTV-6 live-vaccine-strain. Although most of the other genome segments also showed very high levels of identity to the BTV-6 vaccine (99.7 to 100%), Seg-10 showed greatest identity (98.4%) to the BTV-2 vaccine (RSAvvv2/02), indicating that NET2008/05 had acquired a different Seg-10 by reassortment. Although Seg-7 from NET2008/05 was also most closely related to the BTV-6 vaccine (99.7/100% nt/aa identity), the Seg-7 sequence derived from the blood sample of the same animal (NET2008/06) was identical to that of the Netherlands BTV-8 (NET2006/04 and NET2007/01). This indicates that the blood contained two different Seg-7 sequences, one of which (from the BTV-6 vaccine) was selected during virus isolation in cell-culture. The predominance of the BTV-8 Seg-7 in the blood sample suggests that the virus was in the process of reassorting with the northern field strain of BTV-8. Two genome segments of the virus showed significant differences from the BTV-6 vaccine, indicating that they had been acquired by reassortment event with BTV-8, and another unknown parental-strain. However, the route by which BTV-6 and BTV-8 entered northern Europe was not established.

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein Erns and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E rns purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E rns with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E rns genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E rns (amino acid 476 in the polyprotein of CSFV). Replacement of the E rns gene of an infectious DNA copy of C1.1.1 with the E rns genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HSindependent virus to a virus that uses HS as an E rns receptor.

Envelope glycoprotein El (gp5l to gp54) is the most antigenic protein of hog cholera virus or classical swine fever virus (CSFV). Four antigenic domains, A to D, have been mapped on El with a panel of monoclonal antibodies (MAbs) raised against CSFV strain Brescia. The boundaries of these domains have been established by extensive studies on binding of MAbs to transiently expressed deletion mutants of El (P. A. van Rijn, E. J.

Infectious RNA was transcribed for the first time from a full-length cDNA template of the plus-strand RNA genome of a pestivirus. The genome of the C strain, which is a vaccine strain of classical swine fever virus, was sequenced and used to synthesize the template. The cDNA sequence of the C strain was found to be 12,311 nucleotides in length and contained one large open reading frame encoding a polyprotein of 3,898 amino acids. Although there were mostly only small differences between the sequence of the C strain and the published sequences of strains Alfort and Brescia, there was one notable insertion of 13 nucleotides, TTTTCTTTTTTTT, in the 3 noncoding region of the C strain. Furthermore, we showed that the sequences at the 5 and 3 termini of the C strain are highly conserved among pestiviruses. We found that the infectivity of the in vitro transcripts of DNA copies pPRKflc-113 and pPRKflc-133 depended on the correctness of the nucleotide sequence. The in vitro transcripts of pPRKflc-133 were infectious, whereas those of pPRKflc-113 were not. In fact, only 5 amino acids among the complete amino acid sequence determined this difference in infectivity. However, virus FLc-133, which was generated from pPRKflc-133, cannot be differentiated from native C-strain virus. Therefore, we exchanged the region encoding the antigenic N-terminal half of envelope protein E2 in pPRKflc-133 with the equivalent region of strain Brescia. The resulting hybrid virus, FLc-h6, could be differentiated from the C strain and from FLc-133 with monoclonal antibodies directed against envelope proteins E rns and E2 of strain Brescia and the C strain. To be suitable for further vaccine development, viruses generated from pPRKflc-133 should grow at least as well as native C-strain virus. In fact, we found that FLc-133, hybrid virus FLc-h6, and the C strain grew equally well. We concluded that pPRKflc-133 is an excellent tool for developing a classical swine fever marker vaccine and may prove valuable for studying the replication, virulence, cell and host tropism, and pathogenesis of classical swine fever virus.

Passage of native classical swine fever virus (CSFV) in cultured swine kidney cells (SK6 cells) selects virus variants that attach to the surface of cells by interaction with membrane-associated heparan sulfate (HS).Animal experiments indicated that this adaptive Ser-to-Arg mutation had no effect on the virulence of CSFV. Analysis of viruses reisolated from pigs infected with these recombinant viruses indicated that replication in vivo introduced no mutations in the genes of the envelope proteins E rns , E1, and E2. However, the blood of one of the three pigs infected with the S-RT virus contained also a low level of virus particles that, when grown under a methylcellulose overlay, produced relative large plaques, characteristic of an HS-independent virus. Sequence analysis of such a large-plaque phenotype showed that Arg 476 was mutated back to Ser 476 . Removal of HS from the cell surface and addition of heparin to the medium inhibited infection of cultured (SK6) and primary swine kidney cells with S-ST virus reisolated from pigs by about 70% whereas infection with the administered S-ST recombinant virus produced in SK6 cells was not affected. Furthermore, E rns S-ST protein, produced in insect cells, could bind to immobilized heparin and to HS chains on the surface of SK6 cells. These results indicated that S-ST virus generated in pigs is able to infect cells by an HS-dependent mechanism. Binding of concanavalin A (ConA) to virus particles stimulated the infection of SK6 cells with S-ST virus produced in these cells by12-fold; in contrast, ConA stimulated infection with S-ST virus generated in pigs no more than 3-fold. This suggests that the surface properties of S-ST virus reisolated from pigs are distinct from those of S-ST virus produced in cell culture. We postulate that due to these surface properties, in vivo-generated CSFV is able to infect cells by an HS-dependent mechanism. Infection studies with the HS-dependent S-RT virus, however, indicated that interaction with HS did not mediate infection of lung macrophages, indicating that alternative receptors are also involved in the attachment of CSFV to cells.

Since 1998, Bluetongue virus (BTV)-serotypes 1, 2, 4, 9, and 16 have invaded European countries around the Mediterranean Basin. In 2006, a huge BT-outbreak started after incursion of BTV-serotype 8 (BTV8) in North-Western Europe. More recently, BTV6 and BTV11 were reported in North-Western Europe in 2008. These latter strains are closely related to live-attenuated vaccine, whereas BTV8 is virulent and can induce severe disease in ruminants, including cattle. In addition, Toggenburg orbivirus (TOV) was detected in 2008 in Swiss goats, which was recognized as a new serotype of BTV (BTV25). The (re-)emergency of known and unknown BTV-serotypes needs a rapid response to supply effective vaccines, and research to study this phenomenon. Recently, orbivirus research achieved an important breakthrough by the establishment of reverse genetics for BTV1. Here, reverse genetics for two recent BTV strains representing virulent BTV8 and avirulent BTV6 was developed. For this purpose, extensive sequencing of full-genomes was performed, resulting in the consensus sequences of BTV8/net07 and BTV6/net08. The recovery of ‘synthetic BTV’, respectively rgBTV8 and rgBTV6, completely from T7-derived RNA transcripts was confirmed by silent mutations by which these ‘synthetic BTVs’ could be genetically distinguished from wild type BTV, respectively wtBTV6 and wtBTV8. The in vitro and in vivo properties of rgBTV6 or rgBTV8 were comparable to the properties of their parent strains. The asymptomatic or avirulent properties of rgBTV6 and the virulence of rgBTV8 were confirmed by experimental infection of sheep. Reverse genetics of the vaccine-related BTV6 provides a perfect start to develop new generations of BT-vaccines. Reverse genetics of the virulent BTV8 will accelerate research on the special features of BTV8, like transmission by species of Culicoides in a moderate climate, transplacental transmission, and pathogenesis in cattle.

Functional disruption of dendritic cells (DCs) is an important strategy for viral pathogens to evade host defences. Monocytotropic viruses such as classical swine fever virus (CSFV) could employ such a mechanism, since the virus can suppress immune responses and induce apoptosis without infecting lymphocytes. Here, CSFV was shown to infect and efficiently replicate in monocyte-and in bone marrow-derived DCs. Interestingly, the infected DCs displayed neither modulated MHC nor CD80/86 expression. Stimulation of DCs with IFN-a/TNF-a or polyinosinic-polycytidylic acid (pIC) induced phenotypic maturation with increased MHC and CD80/86 expression, both with mock-treated and infected DCs. In addition, the T cell stimulatory capacity of CSFV-infected DCs was maintained both in a polyclonal T cell stimulation and in specific antigen-presentation assays, requiring antigen uptake and processing. Interestingly, similar to macrophages, CSFV did not induce IFN-a responses in these DCs and even suppressed pIC-induced IFN-a induction. Other cytokines including interleukin (IL)-6, IL-10, IL-12 and TNF-a were not modulated. Taken together, these results demonstrated that CSFV can replicate in DCs and control IFN type I responses, without interfering with the immune reactivity. These results are interesting considering that DC infection with RNA viruses usually results in DC activation. INTRODUCTIONClassical swine fever (CSF) is a highly contagious disease of pigs caused by CSF virus (CSFV) and leads to important economic losses worldwide. CSFV together with bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) form the genus Pestivirus within the family Flaviviridae.CSFV is a monocytotropic viral pathogen, which can efficiently evade and compromise the host's immune system. The virus has a high affinity for reticulo-endothelial cells (Cheville & Mengeling, 1969;Ressang, 1973;Susa et al., 1992) causing lymphopenia, thrombocytopenia, coagulation disorders and atrophy of the thymus and bone marrow Pauly et al., 1998; Sanchez-Cordon et al., 2002;Summerfield et al., 2000Summerfield et al., , 2001. Lymphopenia is caused, at least in part, by apoptosis detectable in uninfected lymphocytes Summerfield et al., 1998b). In addition, viable lymphocytes isolated from CSFV-infected pigs do not respond to mitogen stimulation (Pauly et al., 1998;Summerfield et al., 1998b;Van Oirschot et al., 1983). These modulated cells are not infected. Instead, it is the myeloid population, particularly monocytes (Mo) and macrophages (Mw), that contains the early target cell for infection and replication, both in vivo (Ressang, 1973;Gomez-Villamandos et al., 2001;Sanchez-Cordon et al., 2003;Summerfield et al., 2000;Trautwein, 1988) and in vitro (Knoetig et al., 1999). Despite this clear targeting and tropism, no direct evidence has been found of a role for infected Mo and Mw in the observed immunosuppression and death of T lymphocytes (Knoetig et al., 1999).Dendritic cells (DCs) are one of the primary immunological sentinels of the immune system ...

Four antigenic domains (A, B, C and D) on envelope glycoprotein E1 (gp51-54) of hog cholera virus strain Brescia have been specified by using 13 monoclonal antibodies (MAbs) that recognize non-conserved and conserved epitopes. It was shown that the non-conserved epitopes map to the N-terminal half of E1 by analysis of chimeric E1 proteins of strains Brescia and C. Conserved epitopes, however, could not be mapped using this approach. Here we describe mapping of both conserved and non-conserved epitopes on E1 by the use of an extensive set of single and double deletion mutants of E1 of strain Brescia. Deletion mutants were transiently expressed in COS1 cells and analysed by immunostaining with the 13 MAbs directed against strain Brescia and four MAbs directed against strain C. All MAbs bound to the N-terminal half of El, i.e. amino acids 690 to 866 encoded by the sequence of strain Brescia. Domain B and one epitope in domain C are located between residues 690 and 773. Other epitopes in domain C are located on an extended region, i.e. between residues 690 and 800. Conserved epitopes of domain A are mapped between residues 766 and 866, whereas the only non-conserved epitope in this domain is located between residues 766 and 813. Domain D, represented by one MAb, is located in the same region as this nonconserved epitope of domain A, i.e. between residues 766 and 800. The results suggest the presence of two distinct antigenic units on El, one consisting of domains B and C and the other consisting of domain A.