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The use of a vaccine against classical swine fever virus (CSFV) during an outbreak of CSF should lead to a reduction in the horizontal or vertical transmission of CSFV. The reduction of vertical, i.e. transplacental, transmission of a moderate-virulent strain of CSFV from the sow to its offspring was studied in sows vaccinated once or twice with a CSFV E2 subunit vaccine. Two groups of nine sows were vaccinated with one PD95 dose of the E2 subunit vaccine, approximately four weeks before insemination. A third group of nine inseminated sows served as controls. One group of nine sows were vaccinated again at two weeks after insemination. At ten weeks after the primary vaccination, approximately six weeks after insemination, all 27 sows were challenged intranasally with 10(5) TCID50 of a moderate-virulent strain of CSFV, the Van Zoelen strain. The sows were euthanized at five weeks after challenge, and samples from the sows and fetuses were collected for detection of CSFV. All 27 sows were in gestation at the time of slaughter, CSFV was detected in the fetuses of all unvaccinated sows but it was not detected in any of the samples collected from fetuses of the double-vaccinated sows. Virus was however recovered from the fetuses of one out of nine sows vaccinated once. All the sows, except four double-vaccinated sows, developed CSFV Erns antibodies. Transplacental transmission of CSFV was reduced significantly (p <0.001) in all vaccinated sows. When the results from the experiment were extrapolated to a herd level, it could be concluded that, with 95% certainty, approximately 11% (single vaccination) or 0% (double vaccination), confidence intervals of 0.01-0.44 and 0.0-0.30 respectively, of the pregnant sows would still not be protected against vertical transmission of moderate-virulent CSFV. We conclude that vaccination with the CSFV E2 subunit vaccine can reduce the transmission of moderate-virulent strain of CSFV from the sow to its offspring significantly.

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.

A new method of epitope mapping based on chimeric swine vesicular disease (SVD) viruses produced by fusion PCR (polymerase chain reaction). Seven out of 16 neutralising and non-neutralising newly produced monoclonal antibodies (MAbs) discriminated between SVD isolate ITL/1/66 and NET/1/92. Using fusion PCR eight chimeric viruses were produced containing different supplementary pieces of the P1 region of both parent strains. Using these chimeric viruses we were able to map the epitope regions recognised by these seven neutralising and non-neutralising Mabs. This new method, using chimeric viruses produced by fusion PCR, is particularly valuable for the epitope mapping of non-neutralising MAbs.

The development of a classical swine fever (CSF) subunit marker vaccine, based on viral envelope glycoprotein E2, and a companion diagnostic test, based on a second viral envelope glycoprotein E(RNS), will be described. Important properties of the vaccine, such as onset and duration of immunity, and prevention of horizontal and vertical transmission of virus were evaluated. A single dose of the vaccine protected pigs against clinical signs of CSF, following intranasal challenge with 100LD(50) of virulent classical swine fever virus (CSFV) at 2 weeks after vaccination. However, challenge virus transmission to unvaccinated sentinels was not always completely inhibited at this time point. From 3 weeks up to 6 months after vaccination, pigs were protected against clinical signs of CSF, and no longer transmitted challenge virus to unvaccinated sentinels. In contrast, unvaccinated control pigs died within 2 weeks after challenge. We also evaluated transmission of challenge virus in a setup enabling determination of the reproduction ratio (R value) of the virus. In such an experiment, transmission of challenge virus is determined in a fully vaccinated population at different time points after vaccination. Pigs challenged at 1 week after immunization died of CSF, whereas the vaccinated sentinels became infected, seroconverted for E(RNS) antibodies, but survived. At 2 weeks after vaccination, the challenged pigs seroconverted for E(RNS) antibodies, but none of the vaccinated sentinels did. Thus, at 1 week after vaccination, R1, and at 2 weeks, R=0, implying no control or control of an outbreak, respectively. Vertical transmission of CSFV to the immune-incompetent fetus may lead to the birth of highly viraemic, persistently infected piglets which are one of the major sources of virus spread. Protection against transplacental transmission of CSFV in vaccinated sows was, therefore, tested in once and twice vaccinated sows. Only one out of nine once-vaccinated sows transmitted challenge virus to the fetus, whereas none of the nine twice-vaccinated sows did. Finally, our data show that the E(RNS) test detects CSFV-specific antibodies in vaccinated or unvaccinated pigs as early as 14 days after infection with a virulent CSF strain. This indicates that the E2 vaccine and companion test fully comply with the marker vaccine concept. This concept implies the possibility of detecting infected animals within a vaccinated population.

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.

For a recently developed E2 subunit vaccine against classical swine fever (CSF), the reduction in transmission, at different moments after vaccination, was assessed by animal experiments and statistical calculations. Two experiments were performed to estimate the reproduction ratio R. Experiment 1 consisted of three groups and experiment 2 of two groups each of 10 pigs. In four of these groups, all pigs were vaccinated intramuscularly with the vaccine. The pigs in the fifth group remained unvaccinated (control group). After treatment, half of each group was intranasally inoculated with the virulent CSFV strain Brescia. In the vaccine groups, the following vaccination-challenge intervals were applied: 14, 14, 10, and 7 days, respectively. The occurrence of (contact-) infection was determined using the E(rns) ELISA. In the 7-days interval group and in the control group, virus transmission to all contact pigs occurred, indicating R1. Neither in the two 2-week interval groups nor in the 10-day interval group did contact-infections occur. Hence, the estimated R is less than one, which indicates that an epidemic would fade out. Therefore, the E2 subunit vaccine may be an efficacious tool in a control program during an outbreak of CSF as from 10 days after vaccination.

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.

The purpose of this study was to determine the efficacy and stability of an E2 subunit vaccine against classical swine fever virus (CSFV). The vaccine, which contains E2 produced in insect cells by a baculovirus expression vector is a potential marker vaccine, as it allows discrimination between infected and vaccinated pigs. Several vaccination-challenge experiments were performed to determine the dose that protects 95% of the vaccinated pigs (PD95), and to determine the stability and efficacy of the vaccine several months after production. A single vaccination with a vaccine dose of 32 microg E2 - the estimated PD95 - in a water-oil-water adjuvant prevented clinical signs and mortality due to a CSFV challenge-inoculation three weeks after vaccination. Moreover, virus transmission to susceptible sentinel pigs was prevented in nearly all groups of pigs vaccinated with this dose. The vaccine was stable for at least 18 months, and retained its full potency. These findings indicate that the E2 marker vaccine merits further evaluation for suitability for use in a control program during an outbreak of CSF.

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 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.

A plasmid containing a full-length cDNA copy of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was constructed. When RNA that was transcribed in vitro from this full-length cDNA clone was transfected to BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. The infectious clone of LV enables us to mutagenize the viral genome at specific sites and thus will be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

The 5'-terminal end of the genomic RNA of the Lelystad virus isolate (LV) of porcine reproductive and respiratory syndrome virus was determined. To construct full-length cDNA clones, the 5'-terminal sequence was ligated to cDNA clones covering the complete genome of LV. When RNA that was transcribed in vitro from these full-length cDNA clones was transfected into BHK-21 cells, infectious LV was produced and secreted. The virus was rescued by passage to porcine alveolar lung macrophages or CL2621 cells. When infectious transcripts were transfected to porcine alveolar lung macrophages or CL2621 cells, no infectious virus was produced due to the poor transfection efficiency of these cells. The growth properties of the viruses produced by BHK-21 cells transfected with infectious transcripts of LV cDNA resembled the growth properties of the parental virus from which the cDNA was derived. Two nucleotide changes leading to a unique PacI restriction site directly downstream of the ORF7 gene were introduced in the genome-length cDNA clone. The virus recovered from this mutated cDNA clone retained the PacI site, which confirmed the de novo generation of infectious LV from cloned cDNA. These results indicate that the infectious clone of LV enables us to mutagenize the viral genome at specific sites and that it will therefore be useful for detailed molecular characterization of the virus, as well as for the development of a safe and effective live vaccine for use in pigs.

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.

The primary aim of a bovine virus diarrhea virus (BVDV) vaccine is to prevent transplacental transmission of virus. E2 genes of three BVDV strains, belonging to antigenic groups IA, IB and II, were expressed in insect cells. Three groups of 12 ewes were immunized twice with one of the E2 proteins. A fourth group served as a control. The ewes were served and the pregnant ewes of each vaccination group were allotted to three different challenge groups. Seven weeks after the second vaccination the ewes were challenged intranasally with one of the three BVDV strains. Three weeks later the fetuses were removed and fetal organs were collected for virus isolation. At the day of challenge all vaccinated ewes had neutralizing antibodies against the homologous BVDV strains. One E2 subunit vaccine prevented fetal infection after homologous challenge.

Pure preparations of envelope glycoproteins E(rns) and E2 of classical swine fever virus (CSFV) synthesized in insect cells were used to study infection of porcine and bovine cells with the pestiviruses CSFV and bovine viral diarrhoea virus (BVDV). Almost 100% inhibition of infection of porcine kidney cells with CSFV was produced by 100 microg/ml E(rns). After removal of the virus no E(rns) was needed in the overlay medium (growth medium) to maintain this level of inhibition. In contrast, 100% inhibition of infection of porcine kidney cells with CSFV by 10 microg/ml E2 was only achieved when E2 was added to the overlay medium. When E2 was omitted, a maximum of 50% inhibition was achieved. This indicated that after the virus and E2 were removed from the cells, infection still occurred, by virus particles which were still bound to the cell surface. Treatment with 100 microg/ml E(rns) released these particles from the cell surface. Furthermore, E(rns) bound irreversibly to the surface of cells susceptible or unsusceptible to pestivirus infection and cell-to-cell spread of CSFV was completely inhibited by E2 but not by E(rns). These results demonstrated that E(rns) and E2 interacted with different cell surface receptors. Inhibition of BVDV infection of porcine and bovine cells by CSFV E2 suggested that CSFV E2 and BVDV E2 share an identical receptor. BVDV strain 5250 isolated from pigs was efficiently inhibited by CSFV E(rns), whereas several BVDV strains isolated from cattle were not, suggesting that the conformation of E(rns) plays a role in host tropism.

Sensitive methods are required to study the early pathogenesis of swine vesicular diseases (SVD). Therefore, two new methods, immunohistochemistry (IHC) and in-situ hybridization (ISH), were developed and tested for their specificity and sensitivity. With these methods the SVD virus (SVDV) infection in cytospins of primary porcine kidney cells and in frozen skin sections was investigated. Both IHC and the ISH showed a specific cytoplasmic staining, but the IHC detected more infected cells than the ISH. Furthermore, both IHC and ISH were able to detect SVDV in skin sections 4.5 h after infection. It is concluded that IHC is the most suitable and simplest method to identify cells and tissues that support the initial replication of swine vesicular disease virus. However, IHC can only be applied to frozen sections, whereas ISH can also be used in paraformaldehyde-fixed tissues.

Conventionally, the genus Pestivirus of the family Flaviviridae has 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 the Pestivirus genus. Four groups correspond to defined genotypes, whereas the two other groups could be new genotypes within the Pestivirus genus. 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., (Copyright 1997 Academic Press.)

Classical swine fever virus and bovine virus diarrhea virus are members of the genus pestivirus, which belongs to the family of the Flaviviridae. Recently, envelope glycoprotein Erns was identified as an RNase. RNases can express different biological actions. They have been shown to be neurotoxic, antihelminthic, and immunosuppressive. We studied the immunosuppressive properties of Erns in vitro. The glycoprotein totally inhibited concanavalin A-induced proliferation of porcine, bovine, ovine, and human lymphocytes. We then studied the direct cytotoxic effects of Erns on lymphocytes and epithelial cells in protein synthesis assays. Erns strongly inhibited the protein synthesis of lymphocytes of different species, without cell membrane damage. This suggested an apoptotic process, and indeed apoptosis of lymphocytes was detected after incubation with Erns. Pestivirus infections are characterized by leukopenia and immunosuppression. Our results suggest that Erns plays an important role in the pathogenesis of pestiviruses.

Lelystad virus (LV), the prototype of porcine reproductive respiratory syndrome virus, is a small enveloped virus, containing a positive strand RNA genome of 15 kb. LV is tentatively classified in the family Arteriviridae, which consists of lactate dehydrogenase-elevating virus (LDV), equine arteritis virus (EAV) and simian hemorrhagic fever virus (SHFV). These viruses have a similar genome organization and replication strategy as coronaviruses, but the size of the genome is much smaller (12-15 kb) and they have different morphological and physicochemical properties. The genome of LV contains eight open reading frames (ORFs) that encode the replicase genes (ORFs 1a and 1b), envelope proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF7). Genomic comparison of European and North American isolates has shown that the structural proteins encoded by ORFs 2 to 7 vary widely. The amino acid sequences of ORFs 2 to 7 of North American strains share only 55 to 79% identical amino acids with those of European strains. Using polyvalent porcine anti-LV serum, gene-specific anti-peptide sera and monoclonal antibodies, we have identified six structural proteins of LV and their corresponding genes. These are: the 15 kDa unglycosylated nucleocapsid protein (N) encoded by ORF7, an 18 kDa unglycosylated integral membrane protein M encoded by ORF6, a 25 kDa N-glycosylated protein encoded by ORF5, a 31-35 kDa N-glycosylated protein encoded by ORF4, a 45-50 kDa N-glycosylated protein encoded by ORF3 and a 29-30 kDa N-glycosylated protein encoded by ORF2. A nomenclature for these structural proteins is proposed.

Envelope glycoprotein E2, formerly called E1 or gp51-54, of classical swine fever virus (CSFV) expressed in insect cells protects swine from classical swine fever. Monoclonal antibodies directed against epitopes of domains B and C and subdomain A1 are neutralizing. The domains are located on two structural antigenic units in a proposed model of the antigenic structure of E2. One unit consists of nonconserved antigenic domains B and C and the other contains highly conserved antigenic domain A. We produced several mutant E2 proteins by use of the baculovirus expression system. Two selected mutants were E2 proteins in which one of the two structural antigenic units, unit B/C or unit A, was deleted. The protective capacity of the mutant E2 proteins was investigated in an immunization experiment in pigs. Titres of the neutralizing responses in pigs immunized with mutant E2 proteins were all comparable with that of intact E2. These vaccinated pigs were protected against an intranasal lethal CSFV challenge, indicating that the immune response induced by one structural antigenic unit of E2 can protect pigs against classical swine fever.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.

To study the efficacy and safety of bovine virus diarrhea virus (BVDV) vaccines there is need for a valid challenge model. We investigated whether sheep can be used in such a challenge model. We intranasally inoculated six groups (A-F) of seronegative sheep at day 49 of gestation with either of five antigenically different BVDV strains and one border disease virus strain. A seventh group (G) was housed for 10 days with a persistently infected calf and an eighth group (H) served as control. From each group half of the sheep were killed at 2 weeks, and half at 4 weeks after infection. For virus isolation five organs were collected from the sheep and seven from the fetuses. All sheep of groups A and H remained seronegative in the ELISA and in the serum neutralization test. At 2 and 4 weeks after infection virus was isolated from almost all fetal organs in six groups. In group A and in the control group no virus was isolated from the fetal organs. The virus distribution patterns in fetuses from sheep housed with the persistently infected calf or intranasally inoculated with the same strain were similar. We concluded that (i) antigenically different BVDV strains can induce congenital infection in sheep and that (ii) the consequences of a contact infection were similar to those after intranasal infection. In a second experiment we infected two groups of seronegative sheep with one of the strains used in the first experiment, before mating. A control group was left uninfected. The sheep were served and all sheep were challenged with antigenically homologous or heterologous BVDV at day 49 of pregnancy. Three weeks after challenge, sheep were killed and the procedure as in the first experiment was followed. None of the fetuses sheep were virus positive whereas all fetuses of the control sheep were virus positive. Hence, the immune response after BVDV infection protects fetuses against homologous and heterologous infection during pregnancy. Sheep may therefore be used in vaccination-challenge experiments to evaluate BVDV vaccine efficacy in preventing congenital infection.

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.

The glycoprotein E (gE) locus in the genome of pseudorabies virus (PRV) was used as an insertion site for the expression of glycoprotein E1 of classical swine fever virus (CSFV). Transcription of E1 in the recombinants M401, M402 or M403 was regulated by the gD promoter of PRV, the immediate early gene promoter of human cytomegalovirus, or the gE promoter of PRV, respectively. Groups of four pigs were vaccinated once intramuscularly with 10(6) plaque forming units (p.f.u.) of the recombinant viruses and challenged intranasally with 100 50% lethal doses of virulent CSFV and with 10(5) p.f.u. of virulent PRV. All pigs vaccinated with M402 were fully protected against both classical swine fever and pseudorabies.

The ORF7 gene, encoding the nucleocapsid protein N of Lelystad virus (LV), was inserted downstream of the P10 promoter into Autographa californica nuclear polyhedrosis virus (baculovirus). The resulting recombinant baculovirus, designated bac-ORF7, expressed a 15-kDa protein in insect cells. This protein was similar in size to the N protein expressed by LV in CL2621 cells when it was analyzed on sodium dodecyl sulfate-polyacrylamide gels. The N protein expressed by bac-ORF7 was immunoprecipitated with anti-ORF7 was immunoprecipitated with anti-ORF7 peptide serum, porcine convalescent-phase anti-LV serum, and N protein-specific monoclonal antibodies, indicating that this N protein had retained its native antigenic structure. The recombinant N protein was immunogenic in pigs, and the porcine antibodies raised against this protein recognized LV in an immunoperoxidase monolayer assay. However, pigs vaccinated twice with approximately 20 micrograms of N protein were not protected against a challenge with 10(5) 50% tissue culture infective doses of LV. Experimental and field sera directed against various European and North American isolates reacted with the N protein expressed by bac-ORF7 in a blocking enzyme-linked immunosorbent assay. Therefore, the recombinant N protein may be useful for developing diagnostic assays for the detection of serum antibodies directed against different isolates of LV.

The genome of Lelystad virus (LV), a positive-strand RNA virus, is 15 kb in length and contains 8 open reading frames (ORFs) that encode putative viral proteins. ORFs 2 to 7 were cloned in plasmids downstream of the Sp6 RNA polymerase promoter, and the translation of transcripts generated in vitro yielded proteins that could be immunoprecipitated with porcine anti-LV serum. Synthetic polypeptides of 15 to 17 amino acids were selected from the amino acid sequences of ORFs 2 to 7 and antipeptide sera were raised in rabbits. Antisera that immunoprecipitated the in vitro translation products of ORFs 2 to 5 and 7 were obtained. Sera containing antibodies directed against peptides from ORFs 3 to 7 reacted positively with LV-infected alveolar lung macrophages in the immunoperoxidase monolayer assay. Using these antipeptide sera and porcine anti-LV serum, we identified three structural proteins and assigned their corresponding genes. Virions were found to contain a nucleocapsid protein of 15 kDa (N), an unglycosylated membrane protein of 18 kDa (M), and a glycosylated membrane protein of 25 kDa (E). The N protein is encoded by ORF7, the M protein is encoded by ORF6, and the E protein is encoded by ORF5. The E protein in virus particles contains one or two N-glycans that are resistant to endo-beta-N-acetyl-D-glucosaminidase H. This finding indicates that the high-mannose glycans are processed into complex glycans in the Golgi compartment. The protein composition of the LV virions further confirms that LV is evolutionarily related to equine arteritis virus, simian hemorrhagic fever virus, and lactate dehydrogenase-elevating virus.

To investigate how altered control of expression of the essential immediate-early (IE) gene of pseudorabies virus influences virus replication and virulence, we replaced the IE promoter with the tissue-specific promoters of the bovine cytokeratin IV gene (CKIV), the bovine cytokeratin VIb gene (CKVIb), or the inducible promoter of Drosophila heat shock gene HSP70. We compared expression of the IE gene of the wild-type virus and recombinant viruses in different cell types and at different temperatures and found that IE expression had become cell type or temperature dependent. When a recombinant virus was titrated on nonpermissive cells or was titrated at nonpermissive temperatures in vitro, the plating efficiency was reduced by more than 99%. Mice were inoculated subcutaneously (s.c.), intraperitoneally (i.p.), or intranasally (i.n.) with a dose equal to 100 times the 50% lethal dose of the wild-type virus. After inoculation with temperature-sensitive recombinant N-HSP, two (s.c.), two (i.p.), and four (i.n.) of five mice died. However, at this dose, recombinant N-CKIV, which contains a promoter specific for stratified epithelial tissue of the tongue mucosa, was not lethal when inoculated s.c. or i.p. but killed four mice when inoculated i.n. Recombinant N-CKVIb, which contains a promoter specific for the suprabasal layers of the epidermis, was not lethal after inoculation by any of the three routes. In explant cultures of nasal mucosa of pigs, replication of N-CKIV and N-CKVIb was not markedly reduced in the epithelium. However, in contrast to results obtained with wild-type virus, infection of the stroma was not observed. We conclude that the replicative ability and virulence of pseudorabies virus can be influenced by altering control of expression of the IE gene.

We studied in vivo recombination between a thymidine kinase (TK) negative, glycoprotein E (gE) negative, attenuated strain and a virulent strain of pseudorabies virus (PRV) in pigs. To simplify the detection of recombination we inserted different but overlapping (375 bp) parts of the E1 gene of classical swine fever virus into the gG locus of both virus strains. Recombination between the E1 sequences of these viruses results in reconstitution of the complete E1 coding sequence and expression of the E1 protein. Since E1 is highly immunogenic, we expected to detect in vivo recombination in co-inoculated pigs by the presence of serum antibodies against E1. However, after co-inoculation of pigs with high doses of both virus strains, we were unable to detect antibodies against E1, suggesting that in vivo recombination did not occur or remained below the detection limit. Analysis of individual progeny viruses showed that 13 out of 995 (1.3%) possessed a recombinant TK-negative gE-positive phenotype. In contrast, no E1-positive viruses were detected among 5000 analyzed. This result showed that in vivo recombination between the two virus strains did occur, but was much more frequent between the TK and gE loci than between the E1 sequences. Similar results were obtained in in vitro recombination experiments in which possible growth differences between the various virus strains were excluded. The different recombination frequencies could not be attributed to the difference in distance of the genetic loci since recombination between mutations at a distance of 266 bp in the TK gene occurred as frequent as recombination between the TK and gE genes which are separated by approximately 60 kilobasepairs. These results indicate that some property of the E1 sequence and/or the location of the E1 sequence within the PRV genome affects the frequency of recombination.

The genome of Lelystad virus (LV), a positive-strand RNA virus, is 15 kb in length and contains 8 open reading frames that encode putative viral proteins. Synthetic polypeptides of 15 to 17 amino acids were selected from the amino acid sequences of ORFs 2 to 7 and anti-peptide sera were raised in rabbits. Using these anti-peptide sera and porcine anti-LV serum, we identified three structural proteins and assigned their corresponding genes. Virions were found to contain a nucleocapsid protein of 15 kDa (N), an unglycosylated membrane protein of 18 kDa (M), and a glycosylated membrane protein of 25 kDa (E). The N protein is encoded by ORF7, the M protein is encoded by ORF6, and the E protein is encoded by ORF5.

We studied in vivo recombination of pseudorabies virus (PRV) by inoculating mice with non-lethal mutants that carry a small deletion or insertion in the thymidine kinase (TK) gene or the ribonucleotide reductase (RR) gene. After co-inoculation of mice with two different mutants, homologous recombination between the viral genomes resulted in the generation of wild-type PRV that was highly lethal for mice. Thus, recombination could easily be assessed by monitoring survival of inoculated animals. Our results demonstrated that recombination was only detectable when high doses of virus were used. Intragenic recombination was more efficient between mutations in the TK gene than between mutations in the RR gene. Efficient intragenic recombination in the TK gene occurred between mutations which were separated by as few as 266 nucleotides. When two mutants were inoculated with an interval of 2 h, recombination still occurred. No recombination could be detected when the viruses were inoculated at the same time but in separate parts of the body. When inoculated separately, none of the mutants tested could be isolated from the brains of mice. Virus could be recovered from the brain, however, after co-inoculation. Surprisingly, of these viruses 36-39% possessed the parental mutant genotype. This observation indicates that complementation enables these mutants to replicate in the brain and suggests that complementation may contribute to pathogenicity of PRV.

Strain 783 of pseudorabies virus (PRV) is a genetically engineered vaccine which contains three deletions. The purpose of this study was to examine the effect of one of the deletions, which until now has not been characterized. The deletion occurs within the inverted repeats. Seventy-one base pairs (bp) were deleted, including one of the repeat sequence elements related to the TAATGARATTC boxes detected within the promoter and enhancer region of the immediate early (IE) genes of herpes simplex virus. The deletion affected neither the transcription of the IE gene nor viral growth in vitro. In our animal experiments, one group of pigs was inoculated with the original strain 783 and another with strain 783 which had had the repeat sequences restored. These two groups were then compared to determine the protective efficacy of the two vaccine strains against PRV infection. The deletion in the inverted repeats does not affect the vaccine properties of PRV strain 783: strain 783, with and without the 71 bp deletion in the repeats, protected pigs equally well.

Three groups of 150 SPF chickens were spray-vaccinated with live Newcastle disease La Sota-type vaccine (clone 30) at one day of age, and another three groups were NDV spray-vaccinated at 10 days of age. In each of the two series of NDV-vaccinated groups, one group also received at day-old 10(5) TCID50 of chicken anaemia virus (CAV) also and another group 10(5) TCID50 of CAV plus a low dose of virulent Marek's disease virus (MDV). After one week, chickens of the groups which had been NDV-vaccinated and CAV-infected at day-old, with or without MDV, showed severe respiratory distress, conjunctivitis, drooping wings and ruffled feathers. After two weeks, wet and inflamed eyes were observed. After three weeks the respiratory problems were overcome, but the entire group showed retarded growth as compared with the group which had received NDV vaccine only. The 'respiratory sounds' were milder in the chickens NDV-vaccinated at 10 days of age, about 10% of the chickens showing retarded growth. Mortality in CAV-infected chickens which had received NDV vaccine at day-old was above 30% at 4 weeks of age, and between 15 and 20% when NDV had been administered at the age of 10 days, and was 5% in the two NDC vaccine control groups. Decreased haematocrit levels were measured in all four CAV-infected groups at 14 days of age. In serum samples collected for 6 weeks at weekly intervals from chickens of the six groups, no differences were observed between HI antibody titres against NDV virus. Thus, dual infection with CAV and live NDV vaccine did not impair the humoral immune response against attenuated Newcastle disease vaccine.

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.

Two regions of amino acids homologous to the ribonuclease catalysis domain of the fungal RNases T2 of Aspergillus oryzae and Rh of Rhizopus niveus and the plant S-glycoproteins of Nicotiana alata are perfectly conserved in the amino acid sequence of the envelope glycoprotein E2 of classical swine fever virus (CSFV). To analyze the functional significance of these conserved sequences, the gene encoding E2 was inserted into the p10 locus of baculovirus and expressed in insect cells. Recombinant virus BacCE2 generated a protein which was similar in size (42 to 46 kDa) to wild-type E2 synthesized in swine kidney cells infected with CSFV. Recombinant E2 was purified by immunoaffinity chromatography from the lysate of cells infected with BacCE2 and assayed for RNase activity. RNase activity coeluted with the E2 fraction, indicating that ribonuclease activity is an inherent property of E2. The ribonuclease-specific activity of the protein fraction containing pure E2 was comparable to that of the N. alata S-glycoproteins.

Lelystad virus (LV) is an enveloped positive-stranded RNA virus, which causes abortions and respiratory disease in pigs. The complete nucleotide sequence of the genome of LV has been determined. This sequence is 15.1 kb in length and contains a poly(A) tail at the 3' end. Open reading frames that might encode the viral replicases (ORFs 1a and 1b), membrane-associated proteins (ORFs 2 to 6) and the nucleocapsid protein (ORF7) have been identified. Sequence comparisons have indicated that LV is distantly related to the coronaviruses and toroviruses and closely related to lactate dehydrogenase-elevating virus (LDV) and equine arteritis virus (EAV). A 3' nested set of six subgenomic RNAs is produced in LV-infected alveolar lung macrophages. These subgenomic RNAs contain a leader sequence, which is derived from the 5' end of the viral genome. Altogether, these data show that LV is closely related evolutionarily to LDV and EAV, both members of a recently proposed family of positive-stranded RNA viruses, the Arteriviridae.

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 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.

The processing and protective capacity of E1, an envelope glycoprotein of hog cholera virus (HCV), were investigated after expression of different versions of the protein in insect cells by using a baculovirus vector. Recombinant virus BacE1[+] expressed E1, including its C-terminal transmembrane region (TMR), and generated a protein which was similar in size (51 to 54 kDa) to the size of E1 expressed in swine kidney cells infected with HCV. The protein was not secreted from the insect cells, and like wild-type E1, it remained sensitive to endo-beta-N-acetyl-D-glucosaminidase H (endo H). This indicates that E1 with a TMR accumulates in the endoplasmic reticulum or cis-Golgi region of the cell. In contrast, recombinant virus BacE1[-], which expressed E1 without a C-terminal TMR, generated a protein that was secreted from the cells. The fraction of this protein that was found to be cell associated had a slightly lower molecular mass (49 to 52 kDa) than wild-type E1 and remained endo H sensitive. The high-mannose units of the secreted protein were trimmed during transport through the exocytotic pathway to endo H-resistant glycans, resulting in a protein with a lower molecular mass (46 to 48 kDa). Secreted E1 accumulated in the medium to about 30 micrograms/10(6) cells. This amount was about 3-fold higher than that of cell-associated E1 in BacE1[-] and 10-fold higher than that of cell-associated E1 in BacE1[+]-infected Sf21 cells. Intramuscular vaccination of pigs with immunoaffinity-purified E1 in a double water-oil emulsion elicited high titers of neutralizing antibodies between 2 and 4 weeks after vaccination at the lowest dose tested (20 micrograms). The vaccinated pigs were completely protected against intranasal challenge with 100 50% lethal doses of HCV strain Brescia, indicating that E1 expressed in insect cells is an excellent candidate for development of a new, safe, and effective HCV subunit vaccine.

During the replication of Lelystad virus in alveolar lung macrophages, a 3'-coterminal nested set of six subgenomic RNAs (RNA2 to RNA7) is formed. These contain a common leader sequence derived from the 5' non-coding region of the genomic RNA. In this study, the sequence of the junction sites, i.e. the sites where the leader sequence joins to the body of the subgenomic RNA, was determined for all six subgenomic RNAs. For each subgenomic RNA, six to nine cDNA clones were isolated by means of reverse transcription and PCR. The nucleotide sequence at the junction site was identical for all eight cDNA clones derived from subgenomic RNA4. However, heterogeneity was observed in the nucleotide sequence surrounding the junction sites of the cDNA clones derived from subgenomic RNAs 2, 3, 5, 6 and 7. This heterogeneity suggests that the fusion of the leader to the body of the subgenomic RNA may be imprecise. The junction sites of the six subgenomic RNAs had a conserved sequence motif of six nucleotides (UCAACC or a highly similar sequence). The distance between the junction site and the translation initiation codon of the downstream open reading frame varied from nine to 83 nucleotides.

The genome of Lelystad virus (LV), the causative agent of porcine epidemic abortion and respiratory syndrome (previously known as mystery swine disease), was shown to be a polyadenylated RNA molecule. The nucleotide sequence of the LV genome was determined from a set of overlapping cDNA clones. A consecutive sequence of 15,088 nucleotides was obtained. Eight open reading frames (ORFs) that might encode virus-specific proteins were identified. ORF1a and ORF1b are predicted to encode the viral RNA polymerase because the amino acid sequence contains sequence elements that are conserved in RNA polymerases of the torovirus Berne virus (BEV), equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), the coronaviruses, and other positive-strand RNA viruses. A heptanucleotide slippery sequence (UUUAAAC) and a putative pseudoknot structure, which are both required for efficient ribosomal frameshifting during translation of the RNA polymerase ORF1b of BEV, EAV, and the coronaviruses, were identified in the overlapping region of ORF1a and ORF1b of LV. ORFs 2 to 6 probably encode viral membrane-associated proteins, whereas ORF7 is predicted to encode the nucleocapsid protein. Comparison of the amino acid sequences of the ORFs identified in the genome of LV, LDV, and EAV indicated that LV and LDV are more closely related than LV and EAV. A 3' nested set of six subgenomic RNAs was detected in LV-infected cells. These subgenomic RNAs contain a common leader sequence that is derived from the 5' end of the genomic RNA and that is joined to the 3' terminal body sequence. Our results indicate that LV is closely related evolutionarily to LDV and EAV, both members of a recently proposed family of positive-strand RNA viruses, the Arteriviridae.

This paper reviews the laboratory investigations that led us to isolate the Lelystad virus and demonstrate that this virus causes mystery swine disease. We describe: 1) isolating the virus from the disease; 2) characterizing the virus as a new enveloped RNA virus; 3) reproducing the disease experimentally with the isolated Lelystad virus; 4) isolating the virus from the experimentally induced disease.

Monoclonal antibodies (MAbs) directed against envelope glycoprotein E1 (gp51-54) of hog cholera virus (HCV) strain Brescia have been shown to recognize four different antigenic domains A, B, C and D. Epitopes of within domain A have mainly been found conserved among HCV strains, whereas epitopes within domains B, C and D are not conserved. We used transiently expressed hybrid E1 genes of HCV strains Brescia and "C" to map the non-conserved epitopes on E1. Epitopes in domains B and C are located within the ultimate N-terminal 104 amino acids. The non-conserved subdomain A3 is most probably located between domains B/C and a hydrophobic region, which is highly conserved between HCV strains Brescia and "C". The conserved subdomains A1 and A2 are probably located in the vicinity and C-terminally of this conserved, hydrophobic region, which is near the centre of the E1 amino acid sequence.

We used the polymerase chain reaction (PCR) technique to detect bovine viral diarrhea virus (BVDV) infections in cattle. Of 120 cattle screened in this study, 29 were scored positive for BVDV with both PCR and conventional virus isolation. Ninety cattle were negative in both assays. One cow was scored positive for BVDV with the PCR but was negative with virus isolation. In dilution experiments PCR analysis was at least 10 times more sensitive than BVDV isolation.

The vaccine efficacy of a genetically engineered deletion mutant strain of pseudorabies virus, strain 783, was compared with that of the conventionally attenuated Bartha strain. Strain 783 has deletions in the genes coding for glycoprotein I and thymidine kinase. In experiment 1, which had a 3-month interval between vaccination and challenge exposure, strain 783 protected pigs significantly (P less than 0.05) better against virulent virus challenge exposure than did the Bartha strain. The growth of pigs vaccinated with strain 783 was not arrested, whereas that of pigs vaccinated with the Bartha strain was arrested for 7 days. Of 8 pigs given strain 783, 4 were fully protected against challenge exposure; none of the pigs given strain Bartha was fully protected. In experiment 2, which had a 3-week interval between vaccination and challenge exposure, the growth of pigs vaccinated with strain 783 was arrested for 3.5 days, whereas that of pigs vaccinated with the Bartha strain was arrested for 6 days. In experiment 3, pigs with moderate titer of maternal antibodies were vaccinated twice IM or once intranasally with either strain 783 or Bartha and were challenge-exposed 3 months after vaccination. Pigs given strain 783 twice IM were significantly (P less than 0.05) better protected than were the other pigs. They had growth arrest of only 6 days, compared with 9 days for pigs of other groups, and shed less virus after challenge exposure. Results of this study indicate that the vaccine based on the deletion mutant strain 783 is more efficacious than is the Bartha strain of pseudorabies virus.

The safety of an Aujeszky's disease virus vaccine based on strain 783, a deletion mutant which does not express glycoprotein I and thymidine kinase, was assessed in pigs, calves and sheep. Four-day-old piglets which were inoculated intranasally and intramuscularly with 10(7) plaque forming units (PFU) developed only slight depression and fever. The virus was transmitted to a sentinel piglet. Six weeks after inoculation, the pigs were injected with high doses of corticosteroids in an attempt to reactivate the vaccine virus. The pigs did not shed Aujeszky's disease virus, did not develop a rise in virus neutralising antibody titres and sentinel pigs remained seronegative to Aujeszky's disease virus. Strain 783 was passaged in two series of three- to five-day old piglets, but after the third and fourth passages virus could no longer be recovered. Pregnant sows were inoculated with 10(7) PFU of virus strain 783 around day 35 or on day 85 of pregnancy, and their fetuses and piglets were assayed for Aujeszky's disease virus and antibodies against Aujeszky's disease virus. No evidence was found for transplacental transmission of the virus. Calves and sheep were given 10(7) PFU of virus strain 783 intranasally or intramuscularly; they survived and did not develop clinical signs of Aujeszky's disease. All the sheep and the calves inoculated intramuscularly developed neutralising antibodies to Aujeszky's disease virus.

Genomic RNA of hog cholera virus (HCV) strain Brescia was cloned and sequenced. The nucleotide sequence was deduced from overlapping cDNA clones and comprises 12,283 nucleotides. We cloned the complete 3' end of the HCV genome, but could not unequivocally prove that the cDNA sequence also completely covers HCV RNA at the 5' end. The HCV genome contained one large open reading frame, which spans the viral plus strand RNA and encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438,300. To identify structural HCV glycoproteins, we prepared rabbit antisera against three synthetic peptides deduced from the sequence. Because one of these antisera reacted with a 51- to 54-kDa glycoprotein (envelope protein E1 of HCV) on Western blot, the genomic position of the sequence encoding gp51-54 could be clearly established. The amino acid sequence of Brescia was compared with that of HCV strain Alfort and that of BVDV strains NADL and Osloss. The degree of homology between the two HCV strains was 93%, and between Brescia and the BVDV strains about 70%. NADL contained an inserted sequence of 90 amino acids that was absent from the sequences of Brescia, Alfort, and Osloss, whereas Osloss contained an inserted sequence of 76 amino acids that was absent from the sequences of Brescia, Alfort, and NADL. Sequences in p80, the most homologous protein among pestiviruses, showed similarity to six sequence motifs found conserved in helicase-like proteins represented by eIF-4A. Furthermore, a trypsin-like serine protease domain detected in p80 of BVDV was also found conserved in HCV, suggesting that pestivirus p80 may be bifunctional.

In an earlier report, we described the construction of the genetically engineered pseudorabies virus strain 2.4N3A which does not express glycoprotein gI. Although this strain showed a strongly reduced virulence in 10-week-old seronegative pigs, it could still cause severe disease or death in 3-day-old piglets. To attenuate the strain further, we constructed mutants with a deletion in the viral thymidine kinase gene. One mutant strain, designated 783, has a deletion of 19 base pairs and was shown to be highly immunogenic and safe for vaccination of pigs against pseudorabies virus.

Hog cholera virus RNA was cloned and sequenced. A single major open reading frame (ORF), encoding an amino acid sequence of 3898 residues, was found in the second reading frame of the sequence of one of the cDNA strands. We demonstrated that the ORF spans the length of the viral sense RNA, which implies that it is translated into a precursor polyprotein. Several properties of this polyprotein, like hydrophobicity, position of putative protease cleavage sites, distribution of N-linked glycosylation sites, distribution of cysteines and distribution of acidic and basic residues are described and discussed.

Vaccination of pigs is widely practised to control Aujeszky's disease (AD). Molecular biological research revealed that several conventionally attenuated virus vaccines harbour deletions in their genomes. The deleted genes are nonessential for virus replication and can be involved in the expression of virulence. These findings have prompted several groups to construct well-characterized deletion mutants of AD virus that do not express either glycoprotein gI, gX or gIII. These mutants have also been rendered thymidine kinase negative. Although data on vaccine efficacy and safety have been published, widely varying test conditions have made it impossible to identify the most efficacious deletion mutant vaccine(s). Vaccination enhances the amount of virus required for infection and reduces, but does not prevent, the shedding of virulent virus and the establishment of latency in pigs infected with virulent AD virus. Therefore, while a vaccination programme will reduce the circulation of virus in the field, it will not eliminate AD virus from pig populations. To eradicate AD, the ability to differentiate infected from vaccinated pigs is crucial. The use of marker vaccines enables us to identify infected pigs in vaccinated populations by detecting antibodies against the protein whose gene is deleted from vaccine strains. The antibody response to gI appears to persist for more than 2 years, and all of about 300 field strains tested so far express gI. The use of vaccines lacking gI in combination with an enzyme linked immunosorbent assay to detect antibodies to gI and culling of gI-seropositive pigs, may help to eradicate AD in countries where vaccination is widely practised.