Search

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.

An intriguing difference between the E2 glycoprotein of CSFV and the other groups of pestiviruses (nonCSFV) is a lack of two cysteine residues on positions cysteine 751 and 798. Other groups of pestivirus are not restricted to one species as swine, whereas CSFV is restricted to swine and wild boar. We constructed chimeric CSFV/BVDV E2 genes based on a 2D model of E2 proposed by van Rijn et al. (van Rijn et al. 1994, J Virol 68, 3934–42) and confirmed their expression by immunostaining of plasmid-transfected SK6 cells. No equivalents for the antigenic units B/C and A were found on E2 of BVDVII. This indicates major structural differences in E2. However, the immunodominant BVDVII domain A, containing epitopes with essential amino acids between position 760–764, showed to be dependent on the presence of the region defined by amino acids 684 to 796. As for the A domain of CSFV, the BVDVII A-like domain seemed to function as a separate unit. These combined domains in E2 proved to be the only combination which was functional in viral background of CSFV C-strain. The fitness of this virus (vflc36BVDVII 684–796) seemed to be reduced compared to vflc9 (with the complete antigenic region of BVDVII).

The pestivirus glycoprotein E(rns), a ribonuclease, is expressed on the surface of virions and in infected cells as a disulfide-linked homodimer. E(rns) is involved in the infection process and its RNase activity is probably involved in viral replication and pathogenesis. The most C-terminal cysteine residue forms an intermolecular disulfide bond with another E(rns) monomer, resulting in an E(rns) dimer. To study the function of dimerisation of E(rns) for viral replication, the cysteine residue at amino acid position 438 was mutated into a serine residue. The mutated C438S gene was cloned into a vector containing an infectious cDNA copy of the CSFV C-strain genome. Using reverse genetics, a mutant virus was generated that only expressed monomeric E(rns), confirming that Cys 438 is essential for homo-dimerization. Characterization of this mutant virus and of a baculovirus-expressed C438S mutant protein indicated that the loss of the dimeric state of E(rns) reduced the affinity of binding of virions and E(rns) to heparan sulphate (HS), the receptor for E(rns) on the cell surface of SK6 cells. This suggests that interaction of virus-bound E(rns) homodimers with membrane associated HS may be a joined action of the two HS-binding domains (one in each monomer) present in the homodimer.

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.

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). A Ser-to-Arg change in the C terminus of envelope glycoprotein E rns (amino acid 476 in the open reading frame of CSFV) is responsible for selection of these HS-binding virus variants (M. M. Hulst, H. G. P. van Gennip, and R. J. M. Moormann, J. Virol. 74:9553–9561, 2000). In this investigation we studied the role of binding of CSFV to HS in vivo. Using reverse genetics, an HS-independent recombinant virus (S-ST virus) with Ser 476 and an HS-dependent recombinant virus (S-RT virus) with Arg 476 were constructed. 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 by 12-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.

Two recombinant classical swine fever (CSF) viruses (Flc2, Flc3) transcribed from a DNA copy of the genome of the Chinese (C) strain, a CSF virus vaccine strain, were characterized in vivo in rabbits and pigs. Rabbits were inoculated intravenously with Flc2 or Flc3, the parent C-strain virus, a biologically cloned C-strain or CSF virus strain Brescia (C.1.1.1). After 24–96 h fever was detected in the rabbits inoculated with the different C-strain viruses. Apart from those in the control group, all the C-strain inoculated rabbits had developed CSF virus neutralizing antibodies 4 weeks later and were protected against a parent C-strain challenge. In the second experiment, pigs were inoculated with the parent C-strain or recombinant C-strain virus (Flc2 or Flc3) and then challenged after 4 weeks with the virulent CSF virus strain Brescia. None of the pigs showed clinical signs of classical swine fever after vaccination or challenge, whereas the control pigs developed clinical signs typical for acute CSF. Pigs inoculated with the different C-strain viruses were not viremic after inoculation or challenge, and CSF virus neutralizing antibodies were detected from day 14 onwards. The results from both experiments demonstrated that the two recombinant viruses had retained the biological and immunogenic properties of the parent C-strain in rabbits and pigs. We conclude that the full-length cDNA of the C-strain can serve as a matrix for further development of a live recombinant CSF virus marker vaccine.

Sixteen classical swine fever virus (CSFV) field isolates from outbreaks of classical swine fever from the period between February 1997 and March 1998 in the Netherlands were sequence analysed. Parts of the 5' noncoding region (5'NCR) and the E1/E2 gene were sequenced after RT-PCR. The obtained sequences were compared with isolates of recent outbreaks in Europe and those of former outbreaks in the Netherlands. Sequence alignment of the 5'NCR region (321 bp) revealed that the isolates of the Dutch outbreak of 1997-1998 were closely linked to an isolate of the CSF outbreak that started in Paderborn, Germany in 1996. A relatively large fragment of the E1/E2 gene of 850 bp, including the antigenic region of E2, which is one of the most variable regions of the CSFV genome, was sequenced to determine whether this region can be used for epidemiology within an epizootic. Epidemiological tracing of transmission of virus was followed, starting from the first isolate and a line of five generations of viruses was analysed. Besides this, new isolates which could not be epidemiologically linked to preceding ones were also characterised. Differences between the isolates of the Dutch outbreak were minor both for the linked as well as for the non-linked isolates, indicating that all isolates have a common origin. Furthermore, our data show for the first time the genetic stability of CSFV even in the highly variable antigenic region of the E2 gene during a major epidemic lasting more than 1 year.

Envelope glycoprotein E2 is the most immunogenic protein of classical swine fever virus (CSFV). In a proposed model of the antigenic structure of E2, the N-terminal half of E2 forms two independent structural antigenic units, A and BC. E2 without transmembrane region (E2-TMR) is expressed and secreted into the medium of insect cells by use of the baculovirus expression system. The immune response induced by E2 protects pigs against CSFV. Recently, we showed that the protective immune response to a homologous CSFV challenge can be induced by a single unit, A or BC, of E2. An indirect blocking ELISA, or complex trapping blocking assay (CTB) based on both units is routinely used worldwide for serological diagnosis of CSFV infections. Here we show that E2-TMR is secreted into the medium as a homodimer. This E2 homodimer was used to develop a CTB detecting antibodies directed against one immunogenic unit of E2. Thus, the protective immune response induced by E2 containing one unit was not detected with a modified CTB based on the other unit, whereas immune responses induced by a variety of low virulent CSFV strains were detected with such a modified CTB. These results indicate that a deletion E2 protein in combination with a modified CTB are feasible as CSF marker vaccine and accompanying differentiating diagnostic test.

A pig pestivirus isolate, strain H, was characterized by using reverse transcription-PCR (RT-PCR) and direct sequencing of the amplicons. A duplication of 74 nucleotides was found at the 5' terminus of the 5' noncoding (NC) region, which was also found in RNA isolates from tonsils from two other pigs from the same farm. When the duplication was omitted, the 5' NC region showed 97.8% similarity to bovine viral diarrhoea virus (BVDV) strain Korevaar and 94% to BVDV strain Osloss. Furthermore, the rearrangement of the 5' NC region of strain H was maintained after passaging in different cell lines and is not common for ruminant-like pestivirus isolated from pigs. Phylogenetic analysis based on the deduced amino acid sequence of the E2 gene of strain H confirmed the findings of the 5' NC region and show that this strain belongs to the BVDVIb subgroup. These results show for the first time rearrangements in the 5' NC region of a pestivirus.

Conventionally, the genusPestivirusof the familyFlaviviridaehas been divided into bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV), and border disease virus (BDV). To date, BDV and BVDV have been isolated from different species, whereas CSFV seems to be restricted to swine. Pestiviruses are structurally and antigenically closely related. Envelope glycoprotein E2 is the most immunogenic and most variable protein of pestiviruses. We cloned E2 genes of many different pestivirus strains, including those from a deer and a giraffe. The E2 genes were transiently expressed, characterized with monoclonal antibodies, sequenced, and compared. Based on these data, we can delineate six major groups within thePestivirusgenus. Four groups correspond to defined genotypes, whereas the two other groups could be new genotypes within thePestivirusgenus. One group comprises CSFV strains isolated from swine. A second group consists of BDV strains Moredun, L83, and X818, which have been isolated from sheep, and strain F from swine. A third group contains strain BD78 from sheep, strain 5250 from swine, and strain 178003 from cattle. On the basis of E2, these viruses are very similar to BVDV strains associated with acute severe outbreaks of bovine viral diarrhea, so-called type 2 BVDV. The fourth group consists of BVDV strains originating predominantly from cattle. This BVDV group can be divided into two subtypes or subgroups BVDV Ia and Ib: BVDV Ia contains viruses from the United States, such as like NADL and Oregon, and some others, such as 150022 and 1138 from Europe. Subgroup BVDV Ib contains strain Osloss and several Dutch isolates. The fifth and sixth “groups” could be proposed as two new genotypes and contain strains Deer and Giraffe, respectively.

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.

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 immuno-staining with the 13 MAbs directed against strain Brescia and four MAbs directed against strain C. All MAbs bound to the N-terminal half of E1, 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 non-conserved epitope of domain A, i.e. between residues 766 and 800. The results suggest the presence of two distinct antigenic units on E1, one consisting of domains B and C and the other consisting of domain A.

Two related classical swine fever virus (CSFV) strain Brescia clones were isolated from blood samples from an infected pig. Virus C1.1.1 is a cell-adapted avirulent variant, whereas CoBrB is a virulent variant. Sequence analysis revealed 29 nucleic acid mutations in C1.1.1, resulting in 9 amino acid substitutions compared to the sequence of CoBrB 476 R. Using reverse genetics, parts of the genomes of these viruses, which contain differences that lead to amino acid changes, were exchanged. Animal experiments with chimeric viruses derived from C1.1.1 and CoBrB 476 R showed that a combination of amino acid changes in the structural and nonstructural regions reduced the virulence of CSFV in pigs. Moreover, the presence of a Leu at position 710 in structural envelope protein E2 seemed to be an important factor in the virulence of the virus. Changing the Leu at position 710 in the CoBrB 476 S variant into a His residue did not affect virulence. However, the 710 His in the C1.1.1/CoBrB virus, together with adaptive mutations 276 R, 476 R, and 477 I in E rns , resulted in reduced virulence in pigs. These results indicated that mutations in E rns and E2 alone do not determine virulence in pigs. The results of in vitro experiments suggested that a high affinity for heparan sulfate of C1.1.1 E rns may reduce the spread of the C1.1.1/CoBrB virus in pigs and together with the altered surface structure of E2 caused by the 710 L→H mutation may result in a less efficient infection of specific target cells in pigs. Both these features contributed to the attenuation of the C1.1.1/CoBrB virus in vivo.

Two live recombinant vaccines (Flc9 and Flc11) against classical swine fever (CSF) were evaluated for their capacity to reduce transmission of virulent CSF virus (CSFV) among vaccinated pigs. In Flc9 the 5' terminal half of the E2 gene of the C-strain, a CSFV vaccine strain, was exchanged with the homologous gene of the bovine viral diarrhoea virus (BVDV) strain 5250, the E(rns) gene was exchanged likewise in the chimeric Flc11 virus. Both recombinant vaccines induce an antibody response in pigs that can be distinguished from that induced after a wild-type CSFV infection. Four experiments were performed to estimate the reproduction ratio R after different vaccination-challenge intervals. Each group consisted of ten pigs [specified pathogen free (SPF) pigs or conventional pigs] that were vaccinated once, intramuscularly, either with Flc9 or Flc11 virus or that were not vaccinated. Vaccinated and susceptible pigs were challenged intranasally with the virulent CSFV strain Brescia or Behring, 1, 2 or 4 weeks after vaccination. Whether contact-pigs became infected was determined using a CSFV specific E2 (Flc9) or E(rns) (FLc11) antibody ELISA. In the unvaccinated control groups, virus secretion started from day 2 to 4 after inoculation and all contact pigs became infected. Contact pigs became infected in the group of pigs (SPF or conventional) vaccinated once with Flc9 virus and challenged 1-, 2- or 4-weeks later. The estimates of the R in the groups challenged at 1-, 2- and 4-weeks after vaccination were 0.38, 0 and 0.75, respectively. Contact infected pigs were not detected (R=0) in any of the groups of pigs, vaccinated with Flc11, only SPF pigs were used. In order to achieve a statistical significance of R within the vaccinated groups each of the experiments has to be repeated at least once. The R of pigs vaccinated with Flc11 virus and challenged at 1- or 2-weeks after vaccination was however significantly lower that the reproduction ratio of the unvaccinated groups (P=0.013). The R of pigs vaccinated with Flc9 virus and challenged at 1 (conventional pigs) or 2 weeks (SPF pigs) after vaccination was significantly lower that the reproduction ratio of the unvaccinated groups (P=0.013). In conclusion, both chimeric viruses Flc9 and Flc11 provided good clinical protection against a challenge with virulent CSFV at 1 or 2 weeks after vaccination. Further experiments should be carried out to study more aspects of the efficacy of these recombinant viruses before they can be used as a marker vaccine under field circumstances.

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.

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E rns 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 HS-independent virus to a virus that uses HS as an E rns receptor.

An SK6 cell line (SK6c26) which constitutively expressed the glycoprotein E rns of classical swine fever virus (CSFV) was used to rescue CSFV E rns deletion mutants based on the infectious copy of CSFV strain C. The biochemical properties of E rns from this cell line were indistinguishable from those of CSFV E rns . Two E rns deletion mutants were constructed, virus Flc23 and virus Flc22. Virus Flc23 encoded only the utmost N- and C-terminal amino acids of E rns (deletion of 215 amino acids) to retain the original protease cleavage sites. Virus Flc22 is not recognized by a panel of E rns antibodies, due to a deletion of 66 amino acids in E rns . The E rns deletion mutants Flc22 and Flc23 could be rescued in vitro only on the complementing SK6c26 cells. These rescued viruses could infect and replicate in SK6 cells but did not yield infectious virus. Virus neutralization by E rns -specific antibodies was similar for the wild-type virus and the recombinant viruses, indicating that E rns from SK6c26 cells was incorporated in the viral particles. Pigs vaccinated with Flc22 or Flc23 were protected against a challenge with a lethal dose of CSFV strain Brescia. This is the first demonstration of trans -complementation of defective pestivirus RNA with a pestiviral structural protein and opens new ways to develop nontransmissible modified live pestivirus vaccines. In addition, the absence of (the antigenic part of) E rns in the recombinant viral particles can be used to differentiate between infected and vaccinated animals.

A new method for the recovery of infectious classical swine fever virus (CSFV) from full-length genomic cDNA clones of the C-strain was developed. Classical reverse genetics is based on transfection of in vitro transcribed RNA to target cells to recover RNA viruses. However, the specific infectivity of such in vitro transcribed RNA in swine kidney cells is usually low. To improve reverse genetics for CSFV, a stable swine kidney cell line was established that expresses cytoplasmic bacteriophage T7 RNA polymerase (SK6.T7). A 200-fold increased virus titre was obtained from SK6.T7 cells transfected with linearized full-length cDNA compared to in vitro transcribed RNA, whereas transfection of circular full-length cDNA resulted in 20-fold increased virus titres. Viruses generated on the SK6.T7 cells are indistinguishable from the viruses generated by the classical reverse genetic procedures. These results show the improved recovery of infectious CSFV directly from full-length cDNAs. Furthermore, the reverse genetic procedures are simplified to a faster, one step protocol. We conclude that the SK6.T7 cell line will be a valuable tool for recovering mutant CSFV and will contribute to future pestivirus research.

Envelope glycoprotein E rns of classical swine fever virus (CSFV) has been shown to contain RNase activity and is involved in virus infection. Two short regions of amino acids in the sequence of E rns are responsible for RNase activity. In both regions, histidine residues appear to be essential for catalysis. They were replaced by lysine residues to inactivate the RNase activity. The mutated sequence of E rns was inserted into the p10 locus of a baculovirus vector and expressed in insect cells. Compared to intact E rns , the mutated proteins had lost their RNase activity. The mutated proteins reacted with E rns -specific neutralizing monoclonal and polyclonal antibodies and were still able to inhibit infection of swine kidney cells (SK6) with CSFV, but at a concentration higher than that measured for intact E rns . This result indicated that the conformation of the mutated proteins was not severely affected by the inactivation. To study the effect of these mutations on virus infection and replication, a CSFV mutant with an inactivated E rns (FLc13) was generated with an infectious DNA copy of CSFV strain C. The mutant virus showed the same growth kinetics as the parent virus in cell culture. However, in contrast to the parent virus, the RNase-negative virus induced a cytopathic effect in swine kidney cells. This effect could be neutralized by rescue of the inactivated E rns gene and by neutralizing polyclonal antibodies directed against E rns , indicating that this effect was an inherent property of the RNase-negative virus. Analyses of cellular DNA of swine kidney cells showed that the RNase-negative CSFV induced apoptosis. We conclude that the RNase activity of envelope protein E rns plays an important role in the replication of pestiviruses and speculate that this RNase activity might be responsible for the persistence of these viruses in their natural host.

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 Erns 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.

Envelope glycoprotein E1 (gp51 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 E1 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 E1 (P. A. van Rijn, E. J. de Meijer, H. G. P. van Gennip, and R. J. M. Moormann, J. Gen. Virol. 74:2053-2060, 1993). In this study, we used neutralizing MAbs of domains A, B, and C to isolate MAb-resistant mutants (MAR mutants) of CSFV strain Brescia and Chinese vaccine strain ("C"). The E1 genes of MAR mutants were cloned in a eukaryotic expression vector, and the effects of MAR mutations on epitopes were studied with a panel of 19 MAbs by immunostaining of COS1 cells transiently expressing these mutant E1s. Except for the MAR mutation Cys-->Arg at position 792, which abolished binding of all MAbs of domains A and D, amino acid substitutions affected only MAbs belonging to the same domain as the MAb used to select the MAR mutant. However, a MAR mutation in a particular domain did not per se abolish binding of all MAbs recognizing that domain. Furthermore, MAR mutants possessed conservative as well as nonconservative amino acid substitutions. To investigate the significance of a secondary structure for the binding of MAbs, all cysteine residues in the N-terminal antigenic part of E1 were mutated to serine. We found that the cysteines at positions 693 and 737 were essential for binding by MAbs of domains B and C, whereas those at positions 792, 818, 828, and 856 appeared to be essential for the binding of most MAbs of domains A and D. These results fully comply with the previously proposed two-unit structure of the N-terminal half of E1. One unit consists of antigenic domains B and C, whereas the other unit consists of the highly conserved domain A and domain D. We conclude that the first six cysteines are critical for the correct folding of E1. A model of the antigenic structure of E1 is presented and discussed.