Katrin Mischkale

Ilona Reimann

J Zemke

P König

Dr Martin Beer
email: martin.beer@fli.bund.de

Characterisation of a new infectious full-length cDNA clone of BVDV genotype 2 and generation of virus mutants

Keywords: Bovine viral diarrhea virus type 2, pestivirus, infectious pestivirus clone

Based on their genomic sequences, two genotypes of Bovine viral diarrhea virus (BVDV) can be differentiated, BVDV type 1 (BVDV-1) and BVDV type 2 (BVDV-2). The complete genomic sequence of the highly virulent BVDV-2 strain 890 was cloned as cDNA to establish the infectious cDNA clone p890FL. In vitro-synthesised full-length RNA of p890FL was transfected into bovine cells and infectious virus could be recovered (v890FL). In vitro, recombinant v890FL showed similar growth characteristics as wild type virus 890WT.

However, infection experiments in calves revealed an attenuation of recombinant v890FL in comparison to the parental isolate. Both leukocytopenia and fever were less pronounced in v890FL-infected calves. Nevertheless, viremia and virus shedding were comparable between recombinant and parental BVDV 890. Furthermore, mutants with partial deletions of the genomic region encoding for the autoprotease N pro (p890ΔN pro ) or the capsid protein (p890ΔC) were constructed and characterised. In order to generate pseudovirions, replicon v890ΔC was efficiently trans-complemented on a helper cell line. In summary, the newly developed construct p890FL represents the first infectious full-length cDNA clone for the BVDV-2 strain 890 and offers a useful tool for further studies on the pathogenesis of BVDV-2 and the development of novel recombinant BVDV-2 specific vaccine candidates.

A c c e p t e d M a n u s c r i p t 3

Introduction

The Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. BVDV is closely related to the classical swine fever virus (CSFV) and the ovine border disease virus (BDV) (Fauquet et al., 2005). The pestiviral genome consists of a single stranded positive-sense RNA with a length of about 12.3 kb. It contains one large open reading frame (ORF), which is flanked by non-translated regions (NTR) on both genome termini. The single ORF is translated into one polyprotein, which is co-and posttranslationally processed into the mature proteins N pro , C, E rns , E1, E2, p7, NS2/3, NS4a, NS4b, NS5a and NS5b by viral and cellular proteases (Collett et al., 1988;[START_REF] Lackner | Temporal modulation of an autoprotease is crucial for replication and pathogenicity of an RNA virus[END_REF][START_REF] Meyers | Molecular cloning and nucleotide sequence of the genome of hog cholera virus[END_REF]. In cell culture, two BVDV biotypes have been described: cytopathogenic (cp) and non-cytopathogenic (ncp). While the cp biotype induces apoptosis and cell death (Zhang et al., 1996), the ncp biotype leads to a persistent infection of cell cultures (Donis and Dubovi, 1987). Since the late 1980s, a new type of BVDV infections with severe thrombocytopenia associated with hemorrhagic syndrome in cattle has been described in Northern America [START_REF] Pellerin | Identification of a new group of bovine viral diarrhea virus (BVDV) strains associated with severe outbreaks and high mortalities[END_REF][START_REF] Rebhun | Thrombocytopenia associated with acute bovine virus diarrhea infection in cattle[END_REF][START_REF] Ridpath | Segregation of bovine viral diarrhea virus into genotypes[END_REF]. In

Europe, first observations of hemorrhagic syndrome associated with BVDV were reported in the early 1990s (Broes et al., 1992;Lecomte et al., 1996;[START_REF] Thiel | Case reports of hemorrhagic diathesis in calves with bovine diarrhea virus infection[END_REF]. Analysis of different isolates resulted in classification of BVDV into genotype 1 and 2. Because of their genetic, antigenetic and phylogenetic marked differences, the isolates mentioned above were classified as BVDV genotype 2. The highly virulent strain 890 was isolated by [START_REF] Ridpath | Segregation of bovine viral diarrhea virus into genotypes[END_REF] Furthermore, vaccination against BVDV-1 provided only partial protection from BVDV-2 infections and most monoclonal antibodies against BVDV-1 failed to detect BVDV-2 [START_REF] Bolin | Serological detection and practical consequences of antigenetic diversity among bovine viral diarrhea viruses in a vaccinated herd[END_REF][START_REF] Ridpath | Segregation of bovine viral diarrhea virus into genotypes[END_REF]. The ncp BVDV-2 strain 890 was the first BVDV-2 to be completely sequenced (GenBank accession no. U18059). In comparison to other ncp pestiviruses the ORF is elongated due to an insertion of 228 nucleotides in the genome A c c e p t e d M a n u s c r i p t 4 segment encoding for the non-structural protein NS2 [START_REF] Ridpath | The genomic sequence of a virulent bovine viral diarrhea virus (BVDV) from the type 2 genotype: detection of a large genomic insertion in a noncytopathic BVDV[END_REF]. Here, we describe the establishment of an infectious BVDV-2 cDNA clone of strain 890 as well as selected deletion mutants, allowing further studies concerning BVDV pathogenesis, replication and immunoprophylaxis.

Materials and Methods

Cells and virus

Bovine oesophageal cells (KOP-R, RIE244), European bison thymus cells (WT-R, RIE758) and interferon incompetent Madin-Darby bovine kidney cells (MDBK, RIE728) were obtained from the collection of cell lines in veterinary medicine at the Federal Institute of Animal Health, Insel Riems (CCLV). KOP-R cells were used for transfection of in vitrotranscribed RNA and growth kinetics analysis. WT-R cells were used to establish the WT-R2 cell line, which in turn, was used in trans-complementation experiments. The interferon incompetent MDBK cells were used for transfection of in vitro-transcribed RNA and propagation of the Npro-deleted virus mutant. Cells were grown in Dulbecco´s Modified Eagle Medium (DMEM) supplemented with 10 % BVDV-free fetal calf serum (FCS).

BVDV-2 wild type strain 890 (v890WT) was kindly provided by H. Hehnen (Bayer AG, Monheim, Germany).

Monoclonal antibodies

For the detection of BVDV proteins, monoclonal antibodies (mab) WB 433 (anti-E rns , CVL, Weybridge), WB210 (IgG1, anti-E rns , CVL, Weybridge), CA1/2 (anti-E2, Institute for Virology, TiHo Hannover), CA34/1/2 (anti-E2, Institute for Virology, TiHo Hannover), and mab-mix WB103/105 (anti-NS3, CVL, Weybridge) were used (Edwards et al., 1988).

Secondary antibody anti-mouse

Construction of the full-length cDNA clone and the deletion mutants

Plasmids were amplified in Escherichia coli DH10B TM cells (Invitrogen) and Escherichia coli MDS42 (kindly provided by G.M. Keil, FLI) (Pósfei et al., 2006), respectively. Plasmid DNA was purified by using Qiagen Plasmid Mini or Midi Kit or with the GFX TM Micro Plasmid Prep Kit (Amersham). Primers used for plasmid construction are presented in table 1, primers for mutagenesis of the plasmid constructs are listed in table 2 (synthesised by MWG-Biotech, Ebersberg, Germany; or OPERON Biotechnologies, Berlin, Germany). Restriction enzyme digestion and cloning procedures were performed according to standard protocols.

Construction of the infectious cDNA clone p890FL is schematically illustrated in figure 1.

Organisation of the capsid protein deletion mutant (p890ΔC) and of the N pro deletion mutant (p890ΔN pro ) is shown in figure 2.

The full-length cDNA clone p890FL was constituted from four PCR fragments. RNA for RT-PCR was extracted from bovine cells infected with the parental virus 890WT using TRIZOL reagent (Gibco-Life Technologies) or RNeasy Mini Kit (Qiagen). Copy DNA was generated by using the SuperScript™III Reverse Transcriptase (Invitrogen) according to the instructions of the manufacturer. RT-PCR was performed by using the One-step RT-PCR Kit (Qiagen) or the SuperScript ™ III One-Step RT-PCR System with Platinum ® TaqDNAPolymerase (Invitrogen) according to the supplier´s protocol. DNA based amplification was done using the Expand High Fidelity PCR System (Roche Molecular Biochemicals).

The four PCR fragments (figure 1) were generated by RT-PCR using the appropriate primers (table 1) and subsequently ligated into the plasmid vector pA (kindly provided by Gregor A c c e p t e d M a n u s c r i p t 6 Meyers, FLI Tübingen). SmaI sites at the 5´end of the subcloned fragment 1 and within fragment 2 (nucleotide position 1978) were mutated by site-directed mutagenesis using the QuickChangeII XL Site-Directed Mutagenesis Kit (Stratagene) and the respective primers listed in table 2. Subsequently, by sequencing of the complete p890FL plasmid with the Genome Sequencer (GS20, Roche/454), in all 8 mismatches compared to the parental virus were detected. Two defects were eliminated by site-directed mutagenesis (1) a frame shift due to the deletion of two nucleotides in the p7 encoding region and (2) a substitution of one amino acid (aa) at the 3´end of the region encoding for the NS5a protein by using the primers 890_ORF and 890_ORF_r, and the primer pair 890_NS5 and 890_NS5_r, respectively.

For partial deletion of the N pro encoding sequence, the plasmid p890FL and a PCR fragment amplified with the primers 890_SalI and 890_Npro_r were cleaved with SalI and SnaBI and ligated to generate the construct p890∆N pro -E2. A second PCR fragment was generated by using the primer pair 890_Npro and 890_SnaBI, cleaved with NotI / SnaBI and cloned into the NotI and SnaBI digested plasmid p890∆N pro -E2 to obtain the deletion mutant p890ΔN pro .

The deletion encompasses nt 422-889 (aa 13-168) excepting the first 12 aa which overlap with the internal ribosomal entry site (IRES) region.

The capsid-deleted replicon p890ΔC contained a deletion of aa 201-243 (nt 986-1114) compared to the parental p890FL. The 32 N-terminal amino acids and the 27 C-terminal aa of the capsid protein, which constitute an essential signalase recognition site and which direct translocation of the envelope proteins into the endoplasmatic reticulum (ER) for further processing of the E rns -E1-E2 polyprotein [START_REF] Rümenapf | Structural proteins of hog cholera A c c e p t e d M a n u s c r i p t 23 virus expressed by vaccinia virus, further characterization and induction of protective immunity[END_REF], were retained. For construction of p890ΔC, a PCR fragment using the primers 890_SalI and 890_Capsid_r was amplified. p890FL and the PCR fragment were cleaved with SalI and SnaBI and ligated to generate the construct p890ΔC-E2. In a second step, a PCR fragment was amplified by using A c c e p t e d M a n u s c r i p t 7 the primer pair 890_Capsid and 890_SnaBI_r. The resulting amplicon and p890ΔC-E2 were digested with NotI / SnaBI and both ligated to establish the deletion mutant p890ΔC.

In vitro transcription and RNA transfection

In vitro transcription of the deletion mutants p890ΔN pro , p890ΔC and the full-length construct p890FL was performed using the T7 RiboMax Large-Scale RNA Production System (Promega) according to the manufactur´s instructions after linearising the plasmids with SmaI.

The amount of RNA was estimated by ethidium bromide staining after agarose gel electrophoresis. For RNA transfection, bovine cells were detached using a trypsin solution, washed twice with phosphate buffered saline without Ca++/Mg++ (PBS-) and mixed with 1-5 µg of in vitro sythesised RNA. Electroporation was done by using the GenePulser transfection unit (Biorad) (two pulses at 850 V, 25 µF and 156 ω).

Immunofluorescence staining

Cell cultures were fixed with 4% paraformaldehyde (PFA) and permeabilised with 0.01 % digitonin (IF staining of NS3) or fixed/permeabilised with 80 % acetone (E rns , E2), and incubated with the appropriate working dilution of the respective antibodies for 30 min. After one washing step with PBS -, cells were incubated with the Alexa 488 -conjugated secondary antibody for 30 min and finally washed. IF was analysed by using a fluorescence microscope (Olympus). strain NADL (Collett et al., 1988), and was inserted into the pcDNA3.1 expression plasmid (Invitrogen) using KpnI and NotI restriction sites. The nucleotide sequence of Syn-ORF had been changed to remove splice sites [START_REF] Schmitt | Expression of bovine viral diarrhoea virus glycoprotein E2 by bovine herpesvirus-1 from a synthetic ORF and incorporation of E2 into recombinant virions[END_REF], but retained the original amino acid sequence of ncp BVDV strain PT810 (GenBank accession no. AY078406). Additionally, the first codon of Syn-ORF was changed to a methionine to allow expression of the polyprotein under the control of the HCMV immediate-early promoter present in pcDNA3.1, and a stop codon was inserted behind the last codon. The resulting construct pcDNA_C-E2 (1 _g) was used to transfect WT-R cells with the SUPERFECT reagent (Qiagen). At 2 days post transfection (p.t.), cell culture medium was changed to DMEM supplemented with 10 % bovine serum and 0.5 mg of geneticin G418 per ml. G418-resistant colonies were isolated, replated several times, and stained for E rns and E2 expression using mab WB210, respectively E2-mix (CA 1/2 and CA34/1/2).

Trans-complementation

In vitro-transcribed RNA of p890∆C was transfected into WT-R2 cells and at 72 h p.t. RNA replication was analysed by IF staining with NS3 specific mabs. Supernatants of transfected cells were harvested and the titre of the pseudovirion progeny v890∆C_trans was determined.

Serial passages of v890∆C_trans were performed on complementing WT-R2 cells as well as on non-complementing KOP-R cells.

Virus titration

Infectious titres were determined for virus stocks as well as for growth kinetics analyses, and A c c e p t e d M a n u s c r i p t 9 after trans-complementation of p890∆C. Cell culture supernatants of v890FL-, v890WT-and 890∆N pro -infected cells were harvested, and supernatants containing the trans-complemented pseudovirions (v890∆C_trans) were collected. After freezing, supernatants were titrated in log10-dilutions on KOP-R cells, and titres were determined as median tissue culture infective dose per ml (TCID 50 /ml).

Growth kinetics

For in vitro growth kinetics, KOP-R cells were infected with the recombinant virus v890FL, v890ΔN pro and with the parental virus v890WT, respectively, at a multiplicity of infection (MOI) of 1. Supernatants were collected at 0, 8, 12, 24, 48, 72 and 96 h post infection (p.i.) and virus titres (TCID 50 /ml) were determined.

Real-time RT-PCR analyses

In order to determine the viral RNA replication levels of v890FL, v890WT, and v890∆N pro , KOP-R cells were infected at an MOI of 1 of the appropriate viruses. At 48 h p.i., supernatants and cells were separately collected and RNA was isolated by using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer´s instructions. Uninfected KOP-R cells were included as test control. In order to minimize the risk of cross contamination, a one step RT-PCR was performed using the QuantiTect™ Probe RT-PCR Kit (Qiagen). According to [START_REF] Hoffmann | Validation of a realtime RT-PCR assay for sensitive and specific detection of classical swine fever[END_REF][START_REF] Hoffmann | A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses[END_REF], 5 µl RNA template were added to a total volume of 25 µl, containing 3.5 µl RNase-free water, 12.5 µl 2×QuantiTect Probe RT reaction-buffer, 2.0 µl panpesti-specific FAM-labeled primer/probe mix and 0.25 µl RTenzyme mix. For quantification of the copy numbers, serially diluted BVDV-DI9-RNA [START_REF] Behrens | Characterization of an autonomous subgenomic pestivirus RNA replicon[END_REF] was used as standard RNA. The following temperature profile was

Animal experiment

Ten Simmentaler breed calves, aged between 6 and 8 month, were shown to be free of BVDV-antibodies and -antigen. Calves were randomly allocated into two groups of five animals each, and inoculated with the recombinant v890FL and the parental 890WT virus, respectively. Inoculation was done intranasally with 2×10 6 TCID 50 in a volume of 2 ml (1ml per nostril). To confirm infectious titres, both viral suspensions were backtitrated on KOP-R cells after inoculation. The animals were housed under identical conditions in two different units and were monitored daily for clinical signs and rectal body temperatures. Blood samples were collected to monitor viremia as well as to evaluate leukocyte and thrombocytes counts.

Nasal swabs were investigated for virus shedding throughout the experiment.

Results

Construction and characterisation of the infectious BVDV-2 cDNA clone p890FL

The full-length clone p890FL was constituted from four PCR fragments assembled in the low copy vector pA [START_REF] Meyers | Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhae viruses from cDNA constructs[END_REF]. At the 5´end, the sequence of the T7 promoter was added to enable in vitro transcription and at the 3´end a SmaI restriction site was introduced for plasmid linearisation (figure 1). First sequence analyses revealed introduction of several mutations into the full-length clone. In comparison to the parental virus a frame shift due to the deletion of two nucleotides in the p7 encoding region and 7 aa substitutions allocated over A c c e p t e d M a n u s c r i p t 11 the ORF were detected, one in Npro (aa 147), two in E rns (aa 392, aa 489), one in E2 (aa 851), one in NS3 (aa 2057) and two in NS5a (aa 2905, aa 3221). Two of the mutations, the frame shift and the aa substitution at position 3221 were eliminated by using site directed mutagenesis. Subsequently, the p890FL cDNA clone was again completely sequenced, resulting in detection of a bacterial insertion at aa position 648, accompanied by duplication of the aa sequence GLR. Sequence analysis of the bacterial insertion showed similarities to the bacterial IS10 element, which can be found in E. coli K-12 strain (data not shown).

Thereupon, we analysed the sequence of the RNA re-isolated from cells infected with v890FL. The sequences of the bacterial insertion into the cDNA clone p890FL were not present in the viral RNA of v890FL. As a consequence, we used the E. coli strain MDS42 [START_REF] Pósfai | Emergent properties of reduced-genome Escherichia coli[END_REF] instead of E. coli strain DH10B for transformation of the new plasmid constructs, due to the engineered genome of E. coli strain MDS42 without any sequences encoding mobile bacterial genetic elements.

In order to generate infectious virus progeny, in vitro-transcribed RNA of p890FL was transfected into KOP-R cells. 72 h p.t., RNA replication could be detected in nearly 100 % of the cells by IF staining using NS3 specific mabs (figure 3). By passaging the transfection supernatant, infectious virus v890FL could be recovered. A stock of the second passage was used for in vitro and in vivo characterisation. The in vitro growth analyses indicated a very similar growth of the recombinant virus v890FL compared to parental virus 890WT at earlier time points, with a reduced final virus titre of v890FL (figure 4). In order to analyse RNA replication levels, we performed real-time RT-PCR analyses, which showed similar RNA replication levels of v890FL and v890WT. In supernatants of infected cells, 10 8 to 10 9 RNA copies per ml were detected, and intracellular levels of viral RNA revealed around 10 2 RNA copies per cell (table 3).

Furthermore, the animal experiment with v890FL and v890WT demonstrated an attenuated A c c e p t e d M a n u s c r i p t 12 phenotype of recombinant v890FL if compared to wild type virus 890WT. However, both animal groups showed clinical signs of a severe BVDV infection like depression, reduced feed intake, mild diarrhea and respiratory symptoms. In addition, all animals exhibited pyrexia with a biphasic elevated body temperature curve, with mean maximum body temperatures of nearly 41 °C for the wild type infected group and 39.7 °C for the group infected with the recombinant virus v890FL (figure 5). Interestingly, in both groups no thrombocytopenia could be observed. A marked leukocytopenia was present in both groups with earlier recovery to pre-infection levels in the v890FL infected animals (figure 6). For each animal, viremia could be detected at days 3 to 7 for the group infected with v890FL, and at days 2 to 10 for the group infected with the parental virus v890WT (figure 7). In addition, nasal virus shedding could be observed from day 2 to 10 p.i. (data not shown). Taken together, we observed a milder clinical course after infection with v890FL, and without 'haemorrhagic syndrome', compared to the BVDV-2 strain 890 [START_REF] Bolin | Differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves[END_REF].

Construction and characterisation of BVDV-2 deletion mutant p890ΔN pro

An N pro autoprotease deletion mutant, p890ΔN pro (figure 2), was constructed on basis of the infectious BVDV-2 clone p890FL by partial deletion of the genomic sequence encoding most of N pro (the first 36 nt overlapping with the BVDV IRES were retained). In order to detect viral replication, in vitro-transcribed RNA of p890ΔN pro was transfected into interferon negative MDBK cells. At 72 h p.t., expression of NS3 could be detected by IF staining in nearly 100 % of the transfected cells (figure 3). Transfection supernatant was passaged and infectious virus progeny v890ΔN pro could be recovered. Growth kinetics on interferon competent KOP-R cells showed approximately 100-fold reduced growth of the deletion mutant (figure 8). Nevertheless, real-time RT-PCR analyses indicated a similar RNA A c c e p t e d M a n u s c r i p t 13 replication level of v890ΔN pro in comparison to v890FL and v890WT (table 3).

Construction, trans-complementation and characterisation of BVDV-2 replicon p890ΔC

Replicon p890ΔC is characterised by a partial deletion of 43 aa within the encoding region for the capsid protein (figure 2). 48 h post transfection of in vitro-transcribed RNA into noncomplementing KOP-R cells, autonomous replication of viral proteins could be detected in nearly 100 % of the transfected cells by IF staining (figure 3), but no infectious virus progeny could be recovered. For packaging of the replicon p890ΔC, we established the new helper cell line WT-R2 derived from the European bison. Like the first available helper cell line PT805 [START_REF] Reimann | Trans-complementation of autonomously replicating Bovine viral diarrhea virus replicons with deletions in the E2 coding region[END_REF], WT-R2 cells stably express a synthetic ORF encoding the BVDV-1 structural genes C-E rns -E1-E2. For trans-complementation, in vitro-transcribed RNA of the replicon p890ΔC was transfected into WT-R2 cells, and 72 h p.t. autonomous virus replication was detected by IF staining (figure 9). Infectious pseudovirions v890ΔC_trans could be recovered from transfection supernatants and were serially passaged on WT-R2 cells (figure 9). However, no passaging was possible on non-complementing KOP-R cells, and no replication competent revertants or pseudo-revertants could be detected.

Discussion

Several pestiviral infectious cDNA clones, including CSFV (Meyers et al., 1996a;[START_REF] Ruggli | Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA[END_REF] and BVDV-1 [START_REF] Mendez | Infectious bovine viral diarrhea virus (strain NADL) RNA from stable cDNA clones: a cellular insert determines NS3 production and viral cytopathogenicity[END_REF][START_REF] Meyers | Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhae viruses from cDNA constructs[END_REF][START_REF] Vassilev | Authentic and chimeric full-length genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts[END_REF], have been described. However, only a single infectious BVDV-2 cDNA clone (strain NY´93C) is published [START_REF] Meyer | Recovery of virulent and RNasenegative attenuated type 2 bovine viral diarrhea viruses from infectious cDNA clones[END_REF]. In addition, an infectious transcript of the BVDV-2 strain 890 was established by Dehan et al. (2005). However, the construction of an infectious cDNA clone of strain 890 failed. This study describes construction and (p890FL) and its application for the development of further mutants (p890∆N pro and p890∆C). Although PCR amplification of the whole genome represents a simplification of the cloning strategy, and has been described for pestiviruses [START_REF] Rasmussen | Direct recovery of infectious pestivirus from a full-length RT-PCR amplicon[END_REF], the generation of a full-length PCR fragment for the strain 890 failed. Therefore, p890FL was constructed on the basis of four PCR fragments, which were assembled into the vector pA [START_REF] Meyers | Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhae viruses from cDNA constructs[END_REF]. In vitro-transcribed RNA of p890FL was transfected into bovine cells, and replication could be demonstrated in nearly 100 % of the cells. Subsequently, infectious virus progeny could be recovered (v890FL) from supernatants of transfected cells (figure 3).

In vitro-characterisation of v890FL showed similar growth kinetics with only slightly reduced virus titres, and a similar RNA replication level compared to the parental virus 890WT (figure 4). In infected animals however, we observed an attenuated phenotype of v890FL compared to v890WT with lower mean body temperatures and a shorter viremia (figure 5, 6 and 7). Up to now, the reason for in vivo attenuation of v890FL is not definitely resolved. In addition, our experiments revealed for the parental virus 890WT altogether milder clinical symptoms compared to BVDV-2 strain US890 [START_REF] Bolin | Differences in virulence between two noncytopathic bovine viral diarrhea viruses in calves[END_REF], which may be e.g. age or breed related.

Attenuation of RNA-viruses recovered from cDNA clones reflects their genetic variability, and has been also described for BVDV-2 before (Dehan et al., 2005;[START_REF] Meyer | Recovery of virulent and RNasenegative attenuated type 2 bovine viral diarrhea viruses from infectious cDNA clones[END_REF].

However, there are some amino acid substitutions in the full-length ORF of p890FL which could possibly account for the in vivo attenuation: two in the E rns , one in the E2, and one in the NS5a encoding sequences. One of the E rns point mutations is located at the C-terminus, and is identical to a mutation in an infectious transcript of BVDV-2 890 described by Dehan et al. (2005), which also showed an attenuated phenotype. The second aa substitution could be found in the middle part of E rns near the RNase motif. RNase activity is important for A c c e p t e d M a n u s c r i p t 15 virulence and pathogenicity of BVDV [START_REF] Magkouras | RNasedependent inhibition of extracellular, but not intracellular, dsRNA-induced interferon synthesis by Erns of pestiviruses[END_REF][START_REF] Meyer | Recovery of virulent and RNasenegative attenuated type 2 bovine viral diarrhea viruses from infectious cDNA clones[END_REF][START_REF] Meyers | Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting E rns RNase and N pro protease[END_REF], and therefore further studies will predominantly focus on the reconstitution of the E rns mutations. In contrast, the aa substitution within E2 and NS5a are not located in previously defined functional regions [START_REF] Johnson | The NS5A protein of bovine viral diarrhoea virus interacts with the alpha subunit of translation elongation factor-1[END_REF][START_REF] Reed | The NS5A/NS5 proteins of viruses from three genera of the family flaviviridae are phosphorylated by associated serine/threonine kinases[END_REF][START_REF] Sapay | NMR structure and molecular dynamics of the in-plane membrane anchor of nonstructural protein 5A from bovine viral diarrhea virus[END_REF].

Furthermore, the cDNA clone p890FL was used for the construction of the deletion mutant p890∆N pro by partial deletion of the genomic sequence encoding a predominant part of N pro .

In contrast to CSFV and HCV, the extension of the IRES into the ORF of BVDV is not defined in detail. In order to ensure full activity of the IRES, the first 12 codons were retained.

However, the minimum coding region essential for full efficacy of the IRES region is still discussed. Recent reports describe for BVDV-1 the preservation of nine to 25 codons downstream of the initial start codon to ensure full IRES activity [START_REF] Moes | The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon[END_REF], and for BVDV-2 [START_REF] Meyers | Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting E rns RNase and N pro protease[END_REF] reported four residual codons as sufficient for acceptable growth in vitro. Furthermore, alignment of BVDV-1 and BVDV-2 protein sequences resulted in 13 out of the first 16 codons which are conserved in the BVDV polyprotein [START_REF] Moes | The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon[END_REF]. For CSFV a similar conservation scheme is described (Moers and Wirth 2007). 17 codons of the N-terminus are required for full activity of the CSFV-IRES (Fletcher et al., 2002). However, it has to be mentioned that preservation of the first 12 codons of the N progene of BVDV-2 strain 890 were sufficient to maintain viral replication, but also resulted in a capsid protein with an amino-terminal extension. The deletion mutant p890∆N pro was able to replicate in vitro, and from supernatants of transfected interferon negative MDBK cells infectious virus progeny v890∆N pro could be recovered. The v890∆N pro virus titres detected in MBDK cells were comparable to the titres of v890FL detected in KOP-R cells (data not shown), indicating that there is no marked influence of the amino-terminal extension of the capsid protein on viral viability and growth in cell culture. Comparison of the in vitro growth A c c e p t e d M a n u s c r i p t 16 kinetics of v890∆N pro , v890FL and v890WT on interferon-competent KOP-R cells revealed an approximately 100-fold reduced growth of the deletion mutant v890∆N pro due to the loss of N pro as an interferon antagonist [START_REF] Gil | The amino-terminal domain of bovine viral diarrhea virus Npro protein is necessary for alpha/beta interferon antagonism[END_REF]. However, our results are in contrast to the non-reduced in vitro growth of a BVDV-2 N pro deletion mutant described by [START_REF] Meyers | Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting E rns RNase and N pro protease[END_REF]. Furthermore, N pro deletion mutants are useful candidates for efficient modified live vaccines against BVDV-1 and BVDV-2 with the potency to induce sterile immunity without the risk of establishing persistent infections (P. König unpublished data; [START_REF] Meyers | Bovine viral diarrhea virus: prevention of persistent fetal infection by a combination of two mutations affecting E rns RNase and N pro protease[END_REF][START_REF] Zemke | Novel BVDV-2 mutants as modified live vaccines[END_REF].

In addition, the BVDV-2 replicon p890∆C, with a partial deletion of the genomic region encoding the capsid protein, was constructed. The N-terminal 32 aa and the 27 C-terminal aa of the capsid protein, which are essential for signalase recognition, translocation of the envelope proteins into the ER, and further processing of the E rns -E1-E2 polyprotein [START_REF] Rümenapf | Structural proteins of hog cholera A c c e p t e d M a n u s c r i p t 23 virus expressed by vaccinia virus, further characterization and induction of protective immunity[END_REF] were retained. In vitro-transcribed RNA of p890∆C was able to replicate autonomously in non-complementing bovine cells, since the structural proteins are not essential for pestiviral RNA replication [START_REF] Behrens | Characterization of an autonomous subgenomic pestivirus RNA replicon[END_REF]. Virus progeny could not be recovered from the supernatant of transfected non-complementing bovine cells. However, infectious virus could be generated by packaging the defective genomes by using a helper virus [START_REF] Kupfermann | Bovine viral diarrhea virus: characterization of a cytopathogenic defective interfering particle with two internal deletions[END_REF] or a helper cell line [START_REF] Reimann | Trans-complementation of autonomously replicating Bovine viral diarrhea virus replicons with deletions in the E2 coding region[END_REF]. For transcomplementation and packaging of the replicon p890∆C we constructed the new helper cell line WT-R2 essentially as described for the helper cells PT805 [START_REF] Reimann | Trans-complementation of autonomously replicating Bovine viral diarrhea virus replicons with deletions in the E2 coding region[END_REF]. The replicon p890∆C was efficiently trans-complemented and packaged into pseudovirions by using WT-R2 cells. Recombination or reversion during generation of the pseudovirions was not observed, and in contrast to experiments with BVDV-1ΔC replicons and PT805 cells [START_REF] Reimann | Trans-complementation of autonomously replicating Bovine viral diarrhea virus replicons with deletions in the E2 coding region[END_REF][START_REF] Reimann | Packaged replicons of bovine viral diarrhea virus are capable of inducing a protective immune response[END_REF], the recombinant WT-R2 cells even allowed the passaging of

  IgG ALEXA 488 (Molecular Probes) was used for A c c e p t e d M a n u s c r i p t 5 immunofluorescence (IF) staining.

2. 6 .

 6 Trans-complementation of the replicon p890ΔC 2.6.1. Establishment of C-E rns -E1-E2 expressing WT-R2 cells The genomic region encoding the structural proteins (C-E rns -E1-E2) of ncp BVDV-1 strain A c c e p t e d M a n u s c r i p t 8 PT810 (Wolfmeyer et al., 1997) was cloned as a chemically synthesised synthetic open reading frame (Syn-ORF, constructed by GeneArt, Regensburg, Germany). It consisted of 2694 nucleotides extending from nucleotide 890 to 3584 of the nucleotide sequence of BVDV

  A c c e p t e d M a n u s c r i p t 10 used: 30 min at 50 °C (reverse transcription), 14 min at 95 °C (inactivation reverse transcriptase/activation Taq polymerase), followed by 40 cycles of 30 sec at 95 °C (denaturation), 30 sec at 57 °C (annealing) and 60 sec at 62 °C (elongation). Identical temperature profiles were used for all real-time RT-PCR runs and fluorescence values were recorded during the annealing steps.

  first infectious full-length cDNA clone of BVDV-2 strain 890

  the established infectious full-length cDNA clone of BVDV-2 strain 890 could enable new insights in viral biology, especially studies of the 228 nt insertion into the NS2 encoding region of the ncp strain 890, and pathogenesis of BVDV-2. Furthermore, the generated viral mutants can be the basis for the generation of novel safe and efficacious BVDV-2 vaccines.

  ., Aldridge, S., Clarke, M.C., McCauley, J.W., 1996. Cell death induced by cytopathic bovine viral diarrhoea virus is mediated by apoptosis. J. Gen. Virol. 77

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Schematic depiction of the deletion mutants p890ΔN pro and p890ΔC based on the

Figure 3 :

 3 Figure 3: IF analysis of bovine cells transfected with in vitro transcribed RNA of p890FL, p890ΔN pro or p890ΔC. In addition, supernatants of transfected cells were passaged on bovine

Figure 4 :

 4 Figure 4: Growth kinetics of the recombinant virus v890FL (broken line) and the parental

Figure 5 :

 5 Figure 5: Mean body temperatures of calves (n=5) after intranasal infection with the

Figure 6 :

 6 Figure 6: Mean leukocyte counts of calves (n=5) following intranasal infection with the

Figure 7 :

 7 Figure 7: Course of viremia in calves infected with the recombinant v890FL (dark grey bars)

Figure 8 :

 8 Figure 8: Growth kinetics of the deletion mutant v890ΔN pro (dotted line) compared with the

Figure 9 :

 9 Figure 9: Trans-complementation studies with replicon p890ΔC and WT-R2 helper cells. A)

  Figure 2:

Table 1 :

 1 nucleotide sequence of PCR primers used for plasmid constructions

	Primer	Sequence (5´to 3´)	Genomic region a
	F1	TTAACCCGGGTAATACGACTCACTATAGTATAC	1-21 (+)
		GAGATTAGCTAAAGT
	F1_r	ATATCCCGGGGCCTATTATCTTGGTGTTTCTTGG 1950-1982 (-)
	F2	ATATCCCGGGAAGTTTGACACCAACGCCGAAG	1976-2007 (+)
		ATGGC
	F2_r	ATATCCCGGGACGCGTTGGCACGAACACGAGC	6569-6598 (-)
		ATGTTGCC
	F3	CGATACGCGTAACATGGCAGTAGAAACAGC	6593-6618 (+)
	F3_r	GTTCTTACTCTCTAGATAACCGGCTGCTCCC	10804-10834 (-)
	F4	GGGAGCAGCCGGTTATCTAGAGAGTAAGAAC	10804-10834 (+)
	F4_r	ATATGAATTCCCCGGGGGGCCGTTAGAGGCATC	12486-12512 (-)
		CTCTAGTC
	890_Npro	ATATGCGGCCGCATCCGATGAAGGGAGTAAGG	890-913 (+)
		GTGCT
	890_Npro_r	ATATTACGTATGCGGCCGCTGTTTTGTATAAAA	381-421 (-)
		GTTCATTTGAAAACAACTCCATGTGCC
	890_Capsid	GGATGCGGCCGCACCTGAATCAAGAAAGAAAT	1115-1136(+)
		TGG
	890_Capsid_r ATATTACGTATGCGGCCGCTTCTGACTCTTTTGG	968-985 (-)
		GGC
	890_SalI	GGACGTCGACAAACTTTGAATTGG	37-60 (+)
	890_SnaBI_r	CCACAGTACGTATTTACCACCCAAC	3508-3532 (-)
	a genomic region of BVDV-2 strain 890 (GenBank accession no. U18059), symbols in
	brackets show the polarity

Table 2 :

 2 PCR primers used for site directed mutagenesis of plasmid constructs

	Primer	Sequence (5´-3´) a

Table 3 :

 3 Results of the real-time RT-PCR analyses of the recombinant viruses v890ΔN pro , v890FL, and the wild type virus v890WT. KOP-R cells were infected at an MOI of 1. 48 h p.i. supernatants and cells were harvest, respectively.

	Virus	Supernatants (RNA copies/ml)	Cells (RNA copies/cell)
	v890∆N pro	10 8.02	10 1.79
	v890FL	10 8.27	10 2.08
	v890WT	10 8.80	10 1.79

Acknowledgements

We thank Gabriela Adam and Doreen Reichelt for excellent technical assistance. This study was financially supported by Intervet Schering-Plough Animal Health (Netherlands).

Conflict of interest statement

None

A c c e p t e d M a n u s c r i p t 18 Broes, A., Wellemans, G., Dheedene, J., 1992. Syndrôme hémorrhagique chez des bovins infectés par le virus de la diarrhée virale bovine (BVD/MD). Ann. Med. Vet. 137, 33-38. Collett, M.S., Larson, R., Gold, C., Strick, D., Anderson, D.K., Purchio, A.F., 1988. Edwards, S., Sands, J.J., Harkness, J.W., 1988. The application of monoclonal antibody panels to characterize pestivirus isolates from ruminants in Great Britain. Arch. Virol. 102,[197][198][199][200][201][202][203][204][205][206] Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L., 2005. Virus Taxonomie.

Eigth Report of the international commitee on taxonomy of viruses. Elsevier Academic Press.

Fletcher, S.P., Ali, I.K., Kaminski, A., Digard, P., Jackson, R.J., 2002


Thomas Bruun Rasmussen

Ilona Reimann

Åse Uttenthal

Immanuel Leifer

Klaus Depner

Horst Schirrmeier

Martin Beer

Generation of recombinant pestiviruses using a full-genome amplification strategy

Keywords: Pestivirus, Flaviviridae, long RT-PCR, full-genome amplicon, infectious clone, pBeloBAC11

come   L'archive ouverte pluridisciplinaire

(5'NTR and 3'NTR), which are important for viral replication. Generation of pestivirus cDNA clones is a general prerequisite for genetic manipulation of their genomes, a process that can be both lengthy and laborious. Several infectious cDNA clones containing complete pestivirus genomes have been described [START_REF] Meyers | Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs[END_REF][START_REF] Moormann | Infectious RNA transcribed from an engineered full-length cDNA template of the genome of a pestivirus[END_REF][START_REF] Ruggli | Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA[END_REF][START_REF] Vassilev | Authentic and chimeric fulllength genomic cDNA clones of bovine viral diarrhea virus that yield infectious transcripts[END_REF][START_REF] Fan | An improved reverse genetics system for generation of bovine viral diarrhea virus as a BAC cDNA[END_REF]. These cDNA clones have provided new options for directed genetic manipulations of pestiviruses. However, construction of these infectious cDNA clones has been hampered due to the relatively large size of the viral RNA genome and because of genetic instability of the cloned cDNA in combination with the plasmid vectors in the bacterial host. Therefore, new strategies are needed to facilitate construction of stable infectious cDNA clones from a wider range of strains.

Generation of infectious cDNA clones can be facilitated by using long RT-PCR for full-genome amplification. This approach has been applied with success to a number of RNA viruses from the Flaviviridae [START_REF] Tellier | Long PCR and its application to hepatitis viruses: amplification of hepatitis A, hepatitis B, and hepatitis C virus genomes[END_REF][START_REF] Gritsun | Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy[END_REF]Zhang et al., 2001). For pestiviruses, the methodology has been used to obtain the entire open reading frame for sequencing studies [START_REF] Jones | A long distance RT-PCR able to amplify the Pestivirus genome[END_REF] as well as for virus rescue (Rasmussen In the present study, we developed an optimized, efficient and streamlined method for the robust amplification of full-length cDNAs of four strains (BVDV-CP7, BDV-Gifhorn, CSFV-C and CSFV-Paderborn). This procedure was followed by the insertion of the BDV and CSFV amplicons into the stable single-copy vector pBeloBAC11 (Wang et al., 1997). This bacterial artificial chromosome (BAC) has been shown to successfully maintain the fulllength cDNAs of Japanese encephalitis virus (Yun et al., 2003) and BVDV-SD1 [START_REF] Fan | An improved reverse genetics system for generation of bovine viral diarrhea virus as a BAC cDNA[END_REF]. Using this procedure, we have generated new full-length cDNA clones of BDV and CSFV, which enlarges the range of cloned pestivirus genomes that can be manipulated.

Materials & Methods

The different viruses used in this study were; BVDV-CP7 from bovine KOP-R cells (10 6 TCID 50 /ml); CSFV-Paderborn from porcine PK15 cells (10 7 TCID 50 /ml; vaccine strain CSFV-C from porcine PK15 cells (10 4.75 TCID 50 /ml); and BDV-Gifhorn from ovine SFT cells (10 6 TCID 50 /ml). Infected cells were grown in Dulbecco's Modified Eagle Medium supplemented with 10% BVDV-free foetal bovine serum at 37°C in a humidified atmosphere containing 5% CO 2 . The total RNA was extracted from the cells using a combined Trizol/RNeasy protocol. This protocol was used to obtain high-quality full-length genomic RNA. Briefly, total RNA was extracted from 1 ml cell supernatant or cell lysate with 3 ml Trizol

5

LS reagent (Invitrogen) and 0.8 ml chloroform. After phase separation the aqueous phase was mixed with an equal volume of 75% ethanol and 0.7 ml of the resulting mix was transferred sequentially to the same RNeasy column (Qiagen). The column was washed according to the manufacturer's protocol.

Total RNA was eluted from the column with 4 times 30 μl nuclease free water and stored at -80°C until use. Pestivirus genomes were amplified from total RNA preparations using long RT-PCR. It was found that the third RNA eluate empirically performed the best in long RT-PCR, presumable due to a higher proportion of full-length genomic RNA in this fraction.

The total RNA was reverse transcribed at 50°C for 90 minutes using SuperScript III reverse transcriptase kit (Invitrogen) and specific cDNA primers (Table 1). For improved long RT-PCR efficiency, the resulting cDNA was subsequently treated with RNAse H to remove the RNA template. Fulllength PCR amplification was performed using primers specific for the 5'NTR and the 3'NTR of each strain (Table 1) with the Accuprime High Fidelity kit (Invitrogen) which consists of a mixture of Taq and proofreading Pyrococcus GB-D DNA polymerases. Reactions containing 2 μl cDNA were then amplified in a final volume of 50 μl using 94ºC for 30 seconds followed by 35 cycles of 94ºC for 15 seconds, 65ºC for 30 seconds and 68ºC for 12 minutes. Samples The full-length amplicons of about 12.3 kb were also digested with NotI and purified using the Nucleotide removal kit (Qiagen). Equal amounts of vector and amplicons were mixed and ligated in 25 μl reactions overnight at 16ºC using DNA ligase for long fragments (Takara Bio) according to the manufacturer's recommendations. The products (1 μl) were electroporated into electrocompetent DH10B T1 phage resistant E.coli (Invitrogen) and transformants harbouring the cloned amplicons were isolated from selective LB plates containing 12.5 μg/ml chloramphenicol. Small scale preparations of DNA from selected transformants were obtained using the BAC miniprep protocol previously described (Warming et al., 2005) and the presence of the full-length pestivirus cDNA was determined following restriction analysis using NotI.

Selected BAC DNAs were digested with NotI, in vitro transcribed using T7 RNA polymerase and the RNA transcripts were tested for infectivity as previously described [START_REF] Reimann | An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus[END_REF][START_REF] Rasmussen | Virulence, immunogenicity and vaccine properties of a novel chimeric pestivirus[END_REF]. Immunostaining of electroporated porcine SK6 cells (CCLV RIE262) was performed with the panpestivirus antibody (C16) targeting the non-structural NS3 proteins [START_REF] Reimann | An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus[END_REF].

Results

Full-length cDNAs corresponding to four different pestivirus strains were successfully amplified in single long RT-PCRs using an optimized version of the full-genome amplification strategy described by [START_REF] Rasmussen | Direct recovery of infectious pestivirus from a full-length RT-PCR amplicon[END_REF]. The 1). The Accuprime High Fidelity polymerase mix increased the specificity and efficiency of the long RT-PCR compared to the Elongase enzyme mix and produced distinct products of the expected size without smears and aberrant amplification products (Fig. 1). High yields (several micrograms) of DNA were obtained in each long RT-PCR. For amplification of complete genomes in a single RT-PCR, specific primers were designed based on nucleotide sequences of the terminal 5'NTR-and 3'NTR-genomic ends (Table 1). The primers for amplification of BDV-Gifhorn were described in [START_REF] Rasmussen | Direct recovery of infectious pestivirus from a full-length RT-PCR amplicon[END_REF]. The primers for BVDV-CP7 [START_REF] Meyers | Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs[END_REF] Accession number BVU63479), and for the CSFV-C (Accession number AY259122) were based on published sequences. Similarly, the 5'NTR primer for CSFV-Paderborn was designed from Accession number AY072924 [START_REF] Oleksiewicz | Determination of the sequence of the complete open reading frame and the 5'NTR of the Paderborn isolate of classical swine fever virus[END_REF] but this sequence lacks approximately 75 nucleotides from the terminus of the 3'NTR. Therefore, the 3'Paderborn reverse primer was designed using a nucleotide alignment of known full-genome CSFV sequences.

Generally, knowledge of less than 50 nucleotides of the terminal sequences from each of the strains was needed for successful amplification of the whole genome.

By incorporating unique NotI restriction sites within the primers (Table 1), the amplicons were designed for subsequent insertion into pBeloBAC11. Full-length amplicons of the two strains CSFV-C and CSFV-Paderborn, and also the BDV-Gifhorn were obtained using the respective NotI 8 primers (Fig. 2). Amplification and cloning of BVDV-CP7 was not attempted by this strategy due to an internal NotI site in the cDNA sequence.

The amplified genomes were digested with NotI and ligated directly into the NotI site in pBeloBAC11. Electroporation of DH10B E.coli cells with the ligated products yielded small and large bacterial colonies (from a few to several hundreds) after incubation for 24 hours at 37ºC. Both small and large colonies were tested for the presence of full-genome amplicons. Generally, two thirds of the tested transformants carried a BAC containing an insert of the expected size of approximately 12.3 kb (Fig. 3). One third of the transformants either carried BACs with smaller inserts, empty BACs or no visible BAC vector.

There was no correlation between the size of the BAC inserts and the size of the bacterial colonies.

Testing of the BAC clones for their ability to generate virus infectivity in cells is ongoing in our laboratories. The preliminary results of this evaluation show that infectious pestiviruses can be rescued from the DNAs generated by direct insertion of full-length genomes into the BAC vector. For example, selected BAC clones harbouring the CSFV-Paderborn full-length cDNA were NotI digested, in vitro transcribed and the RNA transcripts were tested for infectivity by electroporation into porcine cells. RNA derived from one out of two tested BAC clones (pBeloPader10) was shown to efficiently replicate in porcine cells. The cells electroporated with RNA transcripts from pBeloPader10 showed a high number of NS3-positive cells (as judged by immunofluorescence, Fig. 4) 24 hours later and infectious virus could be recovered after 1 st and 2 nd cell passages. In addition, RNA transcripts derived from each of these three BAC clones was found to be replicating in the cells (data not shown). However, only rather few cells were scored as positive after the 1 st cell passage indicating a possible growth defect in these constructs.

Discussion

The use of the full-length genome amplification strategy for amplification of pestivirus genomes has several advantages; (i) the same optimized protocol can be used for amplification of a wide selection of strains; (ii) only limited terminal nucleotide sequence information of a given strain is needed for amplification of the whole genome; (iii) multiple cloning steps can be circumvented since full-length amplicons can be directly in vitro transcribed or inserted into plasmid vectors; and (iv) construction of genetically modified viruses can be facilitated using stable cDNA clones.

In the present study, four different pestiviruses representing each of three main types (CSFV, BVDV and BDV) were amplified by long RT-PCR. This demonstrates that the recently described full-length genome amplification strategy [START_REF] Rasmussen | Direct recovery of infectious pestivirus from a full-length RT-PCR amplicon[END_REF] is of a generic nature since it can be utilized for amplification of diverse pestivirus strains. Furthermore, the direct cloning of the amplified genomes into the stable single-copy BAC vector, pBeloBAC11, shows that we have created an efficient, streamlined strategy for direct preparation of new pestivirus cDNA clones. Full-length cDNAs corresponding to other members of the Flaviviridae, cloned in pBeloBAC11, have previously been shown to be stable after multiple generations in E.coli cells (Yun et al., 2003;[START_REF] Fan | An improved reverse genetics system for generation of bovine viral diarrhea virus as a BAC cDNA[END_REF] and we anticipate that our constructs will display similar stability in the bacterial host but this does need to be determined.

Our new strategy allows construction of stable infectious BAC DNAs from a single full-length PCR product, which gives an increased flexibility in the design of new genetically modified pestiviruses. Since long RT-PCR preserves the genetic variants present in a virus population [START_REF] Chumakov | PCR engineering of viral quasispecies: a new method to preserve and manipulate genetic diversity of RNA virus populations[END_REF] many independent full-length cDNA clones can be obtained and tested which increases the probability of finding infectious constructs. For targeted design of genetically modified pestiviruses, the efforts can now be expedited and focused on, in principal, any pestiviral strain and is hence not limited by the availability of existing infectious cDNAs. Furthermore, our full-genome amplification protocol obviates the time-consuming and costly process of construction and screening of infectious clones by traditional methods. Therefore, the full-genome amplification strategy significantly simplifies and streamlines the workflow for the generation of new recombinant pestiviruses and also facilitates direct fulllength sequence analysis.

  Formatted for Veterinary Microbiology (special ESVV Pestivirus issue) 1 Generation of recombinant pestiviruses using a full-genome amplification strategy Thomas Bruun Rasmussen 1* , Ilona Reimann 2 , Åse Uttenthal 1 , Immanuel Leifer 2 , Klaus Depner 2 , Horst Schirrmeier 2 and Martin Beer 2 1 DTU National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark 2 Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, D-17493 Greifswald-@vet.dtu.dk Introduction The genus Pestivirus is part of the family Flaviviridae and contains economically important animal viruses such as Classical swine fever virus (CSFV), Bovine viral diarrhoea virus (BVDV) and Border disease virus (BDV). Pestivirus virions are enveloped and contain a positive-stranded RNA genome of about 12.3 kb which has a single long open reading frame flanked by non-translated regions

  Formatted for Veterinary Microbiology (special ESVV Pestivirus issue) 4 et al., 2008). Full-length genome amplification facilitates, in addition to virus rescue, direct full-length sequence analysis and the amplicons can also be directly inserted into suitable cloning vectors.

  Formatted for Veterinary Microbiology (special ESVV Pestivirus issue)

( 1 -

 1 10 μl) of each PCR reaction were analysed on a 1 % agarose gel in TBE buffer alongside a 1kb DNA ladder (Fermentas).Standard techniques were used for cloning of the full-length amplicons into pBeloBAC11 (Invitrogen). Briefly, pBeloBAC11 (7507 bp) was digested with NotI and afterwards treated with Antarctic phosphatase (NEB).

  Formatted for Veterinary Microbiology (special ESVV Pestivirus issue) 6

  original protocol was modified by substituting the Elongase enzyme mix with the Accuprime High Fidelity kit. This polymerase mix, in combination with the A c c e p t e d M a n u s c r i p t Formatted for Veterinary Microbiology (special ESVV Pestivirus issue) 7 Superscript III reverse transcriptase, efficiently produced, from each of the different viral RNAs, amplicons of approximately 12.3 kb, corresponding to the full-length genomes (Fig.

  Formatted for Veterinary Microbiology (special ESVV Pestivirus issue)

  Formatted for Veterinary Microbiology (special ESVV Pestivirus issue) 9 from three BAC clones harbouring BDV-Gifhorn full-length cDNA have been tested for infectivity in ovine cells. Following electroporation, viral RNA derived

  Formatted for Veterinary Microbiology (special ESVV Pestivirus issue) 10

  Formatted for Veterinary Microbiology (special ESVV Pestivirus issue) A c c e p t e d M a n u s c r i p t Formatted for Veterinary Microbiology (special ESVV Pestivirus issue) A c c e p t e d M a n u s c r i p t Formatted for Veterinary Microbiology (special ESVV Pestivirus issue) 13 Wang, K., Boysen, C., Shizuya, H., Simon, M.I., Hood, L., 1997. Complete nucleotide sequence of two generations of a bacterial artificial chromosome cloning vector. Biotechniques 23, 992-994. Warming, S., Costantino, N., Court DL, Jenkins, N.A., Copeland, N.G., 2005. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33, e36. Yun, S.I., Kim, S.Y., Rice, C.M., Lee, Y.M., 2003. Development and application of a reverse genetics system for Japanese encephalitis virus. J Virol 77, 6450-6465.Zhang, F., Huang, Q., Ma, W., Jiang, S.,Fan, Y., Zhang, H., 2001. Amplification and cloning of the full-length genome of Japanese encephalitis virus by a novel long RT-PCR protocol in a cosmid vector. J. Virol.Methods 96,[171][172][173][174][175][176][177][178][179][180][181][182]

  is underlined, and NotI sites are shown in italics.b Same primer was also used as cDNA primer.

  Fig. 1. Full-genome amplification of pestiviruses by long RT-PCR using T7-

Fig. 2 .

 2 Fig.2. Full-genome amplification of pestiviruses by long RT-PCR using T7-

Fig. 3 .Fig. 4 .

 34 Fig. 3. NotI digestion of BAC transformants. DNA preparations from BAC

  Figure 1.

Figure 4 .

 4 Figure 4.

Acknowledgements

We are grateful to Gabriela Adam and Doreen Reichelt for technical assistance. We thank Dr. Donata Kalthoff for her valuable advice regarding cloning in BAC vectors. This work was supported by the EU Network of Excellence, EPIZONE (FOOD-CT-2006-016236) and by the Danish Research Council for Technology and Production Sciences (DRCTPS grant 274-07-0198).

Conflict of interest statement

None


H Everett

F J Salguero

S P Graham

F Haines

H Johns

D Clifford

A Nunez

A La Rocca

S Parchariyanon

F Steinbach

T Drew

H Crooke
email: h.crooke@vla.defra.gsi.gov.uk

Characterisation of experimental infections of domestic pigs with genotype 2.1 and 3.3 isolates of classical swine fever virus

Keywords: Classical swine fever, genotypes 2.1 and 3.3, experimental infection

Characterisation of experimental infections of domestic pigs with genotype 2.1 and 3.3 isolates of classical swine

fever virus.

A c c e p t e d M a n u s c r i p t -2 -WH303. Transmission was monitored by use of in-contact animals. Clinical, virological and haematological parameters were observed and an extended macro and histopathological scoring system allowed detailed characterisation of pathological lesions. Infection with the genotype 2.1 isolate resulted in a similar outcome to other recent genotype 2 European strains, whereas the genotype 3.3 strain produced fewer and delayed clinical signs, notably with

Introduction

Although many areas of the world have eradicated classical swine fever (CSF), epizootics in disease-free regions occur regularly and cause considerable financial and sociological impact. The last outbreak in the UK, in 2000, resulted in the destruction of over 73,000 animals. Of these, 31,900 were culled pre-emptively as dangerous contacts, or due to their close proximity to confirmed cases, but were subsequently confirmed as not A c c e p t e d M a n u s c r i p t -3 -infected (Paton, 2002). Early detection is key to ensuring that control measures will minimise the spread and the impact of such devastating diseases [START_REF] Murray | Model framework and principles of emergency management[END_REF]. Identification of CSFV as the etiological agent of some recent outbreaks has taken two months or more after the virus was introduced. For the UK 2000 outbreak, it is estimated that the virus initially entered the index farm in early June, but confirmation of disease did not occur until August [START_REF] Gibbens | Origins of the CSF outbreak[END_REF]. A lack of pathognomonic signs linked to CSF is one of the factors contributing to delays in its recognition and it has been speculated that recent isolates, for which clinical diagnosis on infected farms was difficult, are of lower virulence (Terpstra and de Smit, 2000). Indeed, European isolates from the late 1990's produced less pronounced and delayed signs in experimental inoculations compared to the historical reference strain, Alfort [START_REF] Floegel-Niesmann | Virulence of recent and former classical swine fever virus isolates evaluated by their clinical and pathological signs[END_REF].

The UK 2000 outbreak was caused by a genotype 2.1 CSFV, which is genetically distinct from previous European 2.1 isolates [START_REF] Gibbens | Origins of the CSF outbreak[END_REF].

The origin of this incursion could not be determined. However, it is thought that exposure of outdoor pigs to an illegally imported infected pork product was the most likely source [START_REF] Gibbens | Origins of the CSF outbreak[END_REF]. Other recent outbreaks, that have been caused by genotype 2.1 strains, such as in the Netherlands in 1997 [START_REF] Greiser-Wilke | Molecular epidemiology of a large classical swine fever epidemic in the European Union in 1997-1998[END_REF] and in South Africa in [START_REF] Hoffmann | Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever[END_REF](Sandvik et al., 2005)), highlight that this genotype poses a continued threat to CSF-free countries.

Viruses belonging to genotype 3 have had a more restricted distribution, being predominately constrained to the Far East. Isolates of the sub-genotype 3.3 A c c e p t e d M a n u s c r i p t -4 -have, thus far, only been collected from Thailand. The nucleotide sequences of characterised 3.3 isolates are quite divergent, indicating that this subgenotype may have evolved locally for some time [START_REF] Parchariyanon | Sequence analysis of E2 glycoprotein genes of classical swine fever viruses: identification of a novel genogroup in Thailand[END_REF]. This genetic diversity is also reflected by antigenic heterogeneity: 21 isolates were classified into 7 different antigenic groups based on monoclonal antibody typing. In particular two isolates, CBR/93 and CBR/94/2, did not react with Mab WH303 that, to our knowledge, has previously reacted with all other CSFV strains tested [START_REF] Parchariyanon | Diverisity of classical swine fever virus in Thailand: antigenic and vaccine efficiency studies[END_REF]. This antibody is widely used in differential diagnosis of pestiviruses as it binds an epitope in the E2 glycoprotein, TAVSPTTLR, which is conserved in CSFV but not in BVDV and BDV strains [START_REF] Lin | Deletions of structural glycoprotein E2 of classical swine fever virus strain alfort/187 resolve a linear epitope of monoclonal antibody WH303 and the minimal Nterminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum[END_REF]. The TAVSPTTLR epitope is a virulence determinant, as mutants with amino acid substitutions in this region produce mild clinical signs (Risatti et al., 2006), raising the possibility that viruses like CRB/93 or CBR/94/2 would be particularly difficult to detect.

This study aimed to characterise the clinical, virological, haematological and pathological features associated with infection of pigs with the UK2000/7.1 and CBR/93 isolates. The results add to the knowledge of the behaviour of modern and diverse strains of CSFV in vivo and establish a challenge model for future vaccination experiments. We have also used a modified pathological scoring system to aid the comparison of experimental infections with different strains of CSFV.

Materials and methods

Viruses

A c c e p t e d M a n u s c r i p t -5 -CSFV strain UK2000/7.1 [START_REF] Gibbens | Origins of the CSF outbreak[END_REF] was isolated from a domestic pig during the UK2000 outbreak and passaged three times in cell culture.

CBR/93 was isolated in Thailand in 1993 [START_REF] Parchariyanon | Sequence analysis of E2 glycoprotein genes of classical swine fever viruses: identification of a novel genogroup in Thailand[END_REF]. Viruses were propagated in PK-15 cells as previously described [START_REF] Drew | Classical Swine Fever (hog cholera), In: OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (mammals, birds and bees)[END_REF]. The E2 region of the CBR/93 strain was amplified by PCR using SuperScript III Reverse Transcriptase (Invitrogen) and KOD Hot start DNA polymerase (Novagen) and the nucleotide sequence determined by ABI sequencing.

Animals

Ten-week-old Large White/Landrace cross-breed pigs were obtained from a local commercial supplier. The pigs were clinically healthy and free of antibodies against pestiviruses. All procedures were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986. During the acclimatisation period, biothermal microchips (Destron Technologies Inc.) were implanted subcutaneously behind the left ear for temperature monitoring.

Biochip and rectal temperatures taken at the start of the experiment indicated that biochip temperatures were approximately 1°C lower than rectal temperatures. Throughout the duration of the study, temperatures and clinical signs were recorded twice daily, using a slightly modified version of a scheme described previously [START_REF] Mittelholzer | Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains[END_REF]. The ten parameters, which were scored between 0 and 3, were the same as described except a parameter measuring the leftovers at feed was replaced by a clinical score (CS) for the biochip temperature (37.0°C-38.9°C = CS 0, 39.0°C -39.9°C = CS 1, 40.0°C-40.9°C = CS of 2, 41.0°C or above CS = 3) A c c e p t e d M a n u s c r i p t -6 -

Animal challenge

For each experiment, six pigs were inoculated by intranasal administration of 2ml of CSFV tissue culture supernatant (1ml in each nostril), using a MAD300 nasal drug aerosol delivery device (Wolfe Tory Medical Inc.). Back titration of inocula on PK-15 cells verified that 10 5.6 TCID 50 of both strains was delivered.

After 24h, the inoculated pigs were re-introduced to three in-contact animals in another pen. For each experiment, two uninfected control animals were housed in a separate pen. EDTA blood samples were obtained prior to inoculation and then at 2 to 3 day intervals post infection. Nasal swabs were collected daily. Control animals were sampled at the same time points as inoculated animals. Any animal, with a clinical score above or approaching 15, was euthanized by administration of 20% Pentobarbitol solution, for humane reasons.

Viral RNA quantification in nasal swabs and EDTA blood

Nasal swabs were soaked in 1ml PBS, agitated and then centrifuged at 1500rpm for 7min. Blood samples were collected in EDTA vacutainers (BD Biosciences). Viral RNA was extracted from 140µl nasal swab suspension or EDTA blood using QIAamp 96 DNA Swab BioRobot or QIAamp Viral RNA mini kits (Qiagen), respectively. Viral RNA was quantified using a qRT-PCR one-step Superscript III Platinum kit (Invitrogen). The reaction mix contained 3µl nucleic acid extract, 12.5µl 2x reaction mix, a final concentration of 5mM MgSO 4 , 0.2µl RNAsin, 50nM ROX and 1U Superscript III reverse transcriptase/Platinum Taq mix. Primers CSF100F and CSF192-R [START_REF] Hoffmann | Validation of a real-time RT-PCR assay for sensitive and specific detection of classical swine fever[END_REF] were used at a concentration of 600nM and the probe CSF-A c c e p t e d M a n u s c r i p t -7 -Probe-1 , which was modified with BHQ1 quencher, was used at 200nM. The viral genome copy number was determined by comparison to a ten-fold serial dilution of a standard included in each PCR. This PCR standard consisted of RNA transcribed in vitro (Megashortscript kit, Ambion) from plasmid pCRXLV324-6 which contains the region of the 5'UTR of CSFV strain Alfort 187, flanked by primers V324 and V326 (Vilcek et al., 1994).

Haematology

To monitor thrombocytopenia, EDTA blood (500µl) was centrifuged at 300g for 1min and 5µl plasma were added to 2.5ml MACSQuant Running Buffer (Miltenyi Biotec). Platelets present in 50µl of this suspension were identified based on forward and side scatter characteristics using a MACSQuant flow cytometer (Miltenyi Biotec). Leukocytes present within 50µl of EDTA blood were detected by staining with 10µl of a porcine pan-leukocyte anti-CD45-FITC antibody (AbD Serotec). Samples were then mixed with 940µl FACS lysing solution (BD Biosciences), to ensure lysis of erythrocytes and fixation of cells, prior to assessment of total leukocyte counts in 50µl of the cell suspension using a MACSQuant flow cytometer.

Pathology

Post mortem examinations followed standard operational procedures and any observed lesions were recorded. A macropathology scoring system was established (Table I) using a total of 21 parameters. Each parameter was evaluated from 0 (no lesion) to 3 (severe lesion). Samples were fixed in buffered formalin and embedded in paraffin wax. Sections (4µm) were stained A c c e p t e d M a n u s c r i p t -8 -with H&E for histopathological examination. A histopathological scoring system was also developed using 33 parameters (Table II) and the severity of lesions was also recorded from 0 (no lesion) to 3 (severe). Data from both scoring systems were analyzed by Kruskal-Wallis non-parametric mean comparison test and differences were considered significant for P<0.05.

Results

Characterisation of CSFV CBR/93

To confirm that the CBR/93 isolate is variant at the WH303 epitope, PK-15 cells infected with CSFV strain UK2000/7.1 or CBR/93 were stained and analysed by flow cytometry. Whereas UK2000/7.1 was easily detected with Mab WH303 staining of strain CBR/93 was impaired (data not shown).

Sequence analysis of the E2 region revealed a nucleotide substitution that encodes a serine instead of a proline in the relevant amino acid sequence (TAVSP/STTLR) (Accession number FJ790771).

Clinical features of UK2000/7.1 and CRB/93 infection

Upon inoculation with the UK2000/7.1 strain, two of the animals displayed clinical signs from 7-8 days post inoculation (dpi). The disease then progressed and these two animals were euthanized 13 dpi when their clinical scores were above 15 (Fig 1A). Onset of clinical signs was delayed slightly in the remaining four inoculated animals with scores above 4 occurring at 14 dpi, after which they steadily increased until 17 -18 dpi when the animals were euthanized. The three in-contact animals had initial clinical signs at 16 dpi; the . The most prominent clinical signs were a reddening of the conjunctiva and soft faeces or diarrhoea. These early signs were followed by loss of appetite, lethargy, stiffness of gait and preputial oedema. Notably, no reddening, haemorrhaghes or petechiae of the skin were observed in animals infected with UK2000/7.1 during the duration of the experiment, apart from a diffuse purplish discolouration of the abdominal skin of one of the pigs euthanized on 13 dpi.

In pigs inoculated with the CBR/93 strain, clinical signs were delayed and less severe (Fig 1B). Only one animal had signs resulting in a score above 5 before 15 dpi. The disease then progressed rapidly with two of the animals dying unexpectedly overnight on 18 and 19 dpi and three of the remaining four inoculated animals were euthanized at that point. One pig developed few signs and appeared to be recovering from infection towards the end of the experiment on day 20. Two of the in-contact animals developed mild clinical signs from dpi 18. In addition to the less severe and delayed signs, it was notable that CBR/93 inoculated animals had only a mild, rather insignificant increase in temperature from 14 dpi (Fig 1D). Diarrhoea was again a predominant sign as was lack of attentiveness. With this strain however, we observed a reddening of the skin, particularly of the ears, but very little conjunctivitis. When the results of each group were compared using the macro and histopathological scoring systems no statistical differences were found between UK2000/7.1 and CBR/93 inoculated or UK2000/7.1 and CBR/93 incontact animals. Significant differences were observed between the 3 groups (inoculated, in-contact and control) within the same experiment (P<0.05) (Figure 3).

Viraemia and nasal shedding

Discussion

The clinical signs and outcomes of CSFV infections vary considerably depending on inherent properties of the virus strain as well as host and environmental factors. It is acknowledged that classifying the spectrum of different strains of CSFV into categories of high, moderate or low virulence is difficult. A system for assigning virulence based on the clinical score (CS >15 and temp >41°C as a highly virulent strain, and CS of 5 to 15 as moderately virulent) has been suggested [START_REF] Mittelholzer | Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains[END_REF] and is used by many laboratories. The system is useful to provide comparability between experiments. It is, however, difficult to assign the two strains used in this study according to the parameters of this system. Although two of the animals inoculated with UK2000/7.1 reached scores >15 by 13 dpi, the remaining inoculated or in-contact animals were euthanized before reaching a clinical score of 15 for welfare reasons. However, although these remaining animals may have progressed to reach scores above 15, the course of the disease A c c e p t e d M a n u s c r i p t -13 -was certainly delayed compared to the outcomes described upon infection with strains generally considered as highly virulent such as Brescia, Eystrup [START_REF] Mittelholzer | Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains[END_REF] and ISS/60 [START_REF] Belak | Comparative studies on the pathogenicity and tissue distribution of three virulence variants of classical swine fever virus, two field isolates and one vaccine strain, with special regard to immunohistochemical investigations[END_REF]. The disease seen with the UK2000/7.1 isolate more closely resembled the course of infection observed with recent 2.1 and 2.3 isolates [START_REF] Floegel-Niesmann | Virulence of recent and former classical swine fever virus isolates evaluated by their clinical and pathological signs[END_REF].

Classification of UK2000/7.1 as a moderately virulent strain is therefore appropriate and is consistent with anecdotal observations in the field during the 2000 outbreak that the strain appeared less virulent than historic isolates.

Assessment of the clinical signs caused by CBR/93 in the first 2 weeks would result in the strain being described as having low virulence. However the rapid progression of disease at 17 dpi, resulting in unexpected death of two animals, and a similar degree of pathology at post mortem compared to UK2000/7.1 categorizes this virus in the moderately virulent group.

Importantly, these observations highlight the difficulty in the clinical diagnosis of CSF. In particular clinical signs such as high fever and skin lesions classically associated with the disease may not be present in these early stages.

Although clinical signs were not obvious, high levels of viral RNA were easily detected in both blood and nasal swabs during these early stages of infection. The use of biochips for monitoring temperatures provided practical advantages in terms of ease of monitoring which reduced disturbance of the animals and allowed more frequent measurements. This highlighted the importance of taking temperatures at consistent times as the animals had a marked circadian rhythm. It is possible that hyperthermia would be more apparent with the CBR/93 strain if rectal temperatures were monitored.

However separate assessment of biochip verses rectal temperatures over a longer period indicate that, although the actual values are different, changes in temperature monitored by the two methods correlate well (unpublished data). Despite the differences observed in the clinical signs, infection with both isolates resulted in similar pathological scores. The in-contact animals exposed to both strains were less affected pathologically. This is likely due to a delay in the initial infection although the different dose of infection is also likely to have some effect on the course of disease. The most prominent lesion observed was a generalized lymphadenopathy. Other lesions, such as pinpoint haemorrhages in the kidney or splenic infarcts in the spleen, which are sometimes considered to be pathognomonic of CSF, were observed on only very few occasions. Similar lesions to those observed can be found in animals with PDNS and virulent PRRS, again highlighting the need for early considerations of laboratory testing.

Comparing the virological and haematological parameters many were slightly, although not dramatically, lower and delayed with CBR/93 in comparison with UK2000/7.1. Leukopenia and thrombocytopenia were delayed and the level of viral RNA was lower in the blood during the second week post infection.

A c c e p t e d M a n u s c r i p t -15 -These observations may be linked with the fewer and delayed clinical signs observed with the CBR/93 strain. Preliminary data suggests that there is also a difference in the infection of peripheral blood cells between the two viruses (unpublished data) which may have implications for the clinical outcomes observed. It is also interesting that the mutant virus T3v (Risatti et al., 2006), in which a targeted change replaces the proline in the WH303 epitope, also resulted in delayed clinical signs with reduced viraemia and shedding compared to its' parental strain. This suggests that the delayed and less severe clinical signs observed with CBR/93 may be a result of the changes in the WH303 epitope rather than this being a feature of genotype 3.3 strains in general. However, further studies would be needed with other genotype 3.3 strains to confirm this.

The WH303 epitope on the E2 protein is an immunodominant site, the presence of which is widely utilised as part of the differential diagnosis of CSFV (Zhang et al., 2006). The natural occurrence of CSFV that do not react with this antibody should therefore be heeded during differential diagnosis and highlights that results from procedures reliant on a single CSFV-specific monoclonal antibody must be interpreted with caution. It also raises questions concerning the efficacy of conventional vaccines against such strains.

One of the aims of this study was to establish relevant challenge models for future vaccination and pathogenicity studies. The similarities of the clinical outcome with UK2000/7.1 to other recent genotype 2 isolates and the antigenic and genetic diversity represented by the CBR/93 isolate highlight that these two strains represent relevant challenge models of current CSFV isolates. Both viruses were consistently transmitted to in-contact animals,

  A c c e p t e d M a n u s c r i p t -9 -clinical scores were of a similar, but delayed, profile relative to the inoculated animals. The biochip temperatures correlated well with the clinical scores (Fig 1C)

  In both experiments viral RNA was detected in EDTA blood of all but one of the inoculated animals by 4 dpi, with the viral load increasing to a peak at 11 dpi (Figure2A, B). One of the UK2000/7.1 inoculated animals, which developed early clinical signs, was viraemic by 2 dpi and had a higher viral RNA load than the other UK2000/7.1 inoculated animals. On average the viral RNA copy number in blood was 1 log 10 lower in CBR/93 than UK2000/7.1 inoculated animals between dpi 6 and 11. The pig that developed few clinical signs, following inoculation with CBR/93, became viraemic at the same time as the other CBR/93 inoculated animals, but the viral RNA load did not reach the same level and started to decrease from 11 dpi. In the case of both strains, the in-contact animals became viraemic at 14 dpi, 3-4 days before clinical signs developed (Fig 2A, B).Viral RNA was detected in nasal swabs from all inoculated animals between 5 and 8 dpi; 2-3 days after the animals became viraemic, but before the onset of clinical signs (Fig2A, B). Viral RNA in nasal swabs then increased before reaching a plateau at 14 dpi. The viral load in nasal swabs of CBR/93 inoculated-animals was lower than with UK2000/7.1. Interestingly, low levels of viral RNA were present in the nasal swabs of the in-contact animals shortly after the inoculated animals started shedding, but before these in-contacts became viraemic. This low level persisted, then increased 2 days after the animals became viraemic.HaematologyAnimals, inoculated with either virus, developed thrombocytopenia (Fig 2C, D). Platelet counts decreased below the levels in uninfected control animals A c c e p t e d M a n u s c r i p t -11 -from 6 dpi for UK2000/7.1 inoculated animals, whereas a difference between inoculated and control animals was only observed after 11 dpi for CBR/93 inoculated animals, when the reduction in platelets was then more severe. Animals also developed leukopenia (Fig 2E,F) with leukocyte counts decreasing below levels in control animals from 4 dpi. Pathology Animals inoculated with UK2000/7.1 and CBR/93 isolates had a generalized bilateral lymphadenopathy. The mandibular, lateral and medial retropharyngeal lymph nodes were most affected (Supplemental Figures S1, S2), followed by prescapular and ventral cervical superficial and, to a lesser extent, precrural, ileocaecal and inguinal superficial lymph nodes. Other frequently observed lesions were hydrothorax, hydropericardium, ascitis, secondary bacterial bronchopneumonia and presence of necrotic foci and diphteroid plaques in the ileocaecal valve and colon. Splenic infarcts and petechia in different organs were only observed on very few occasions. The in-contact animals had similar, but less severe lesions, than inoculated animals. Only minimal background lesions were found in control animals. The most common histopathological finding observed in animals inoculated with UK2000/7.1 and CBR/93 strains was a mild to severe lymphoid depletion in the lymph nodes accompanied by hyperemia and haemorrhages. Different degrees of lymphoid depletion were also observed in thymus and spleen. Inflammatory lymphohistiocytic infiltrates were frequently observed as perivascular cuffing in the liver, kidney and encephalon. In the ileocaceal valve and colon, the findings consisted of ulcerative necrotizing colitis withA c c e p t e d M a n u s c r i p t -12 -fibrin deposition. The most commonly observed lung lesions were acute bacterial bronchopneumonia and alveolar and interstitial oedema. The uninfected control animals did not show significant changes.

  Advances in real time RT-PCR and automation of RNA extraction in the last few years open up more possibilities for large-scale and rapid laboratory testing for viral nucleic acid. It would therefore be beneficial if samples were submitted early for laboratory tests, if only to exclude the possibility of CSFV infection.

  ., 2002, The reappearance of classical swine fever in England in 2000, In: Morilla, A., Yoon, K.-J., Zimmerman, J.J. (Eds.) Trends in emerging viral infections of swine. Iowa State Press, Iowa, pp. 153-158. Risatti, G.R., Holinka, L.G., Carrillo, C., Kutish, G.F., Lu, Z., Tulman, E.R., Sainz, I.F., Borca, M.V., 2006, Identification of a novel virulence determinant within the E2 structural glycoprotein of classical swine fever virus. Virology 355, 94-101. Sandvik, T., Crooke, H., Drew, T.W., Blome, S., Greiser-Wilke, I., Moennig, V., Gous, T.A., Gers, S., Kitching, J.A., Buhrmann, G., Bruckner, G.K., 2005, Classical swine fever in South Africa after 87 years' absence. Vet Rec 157, 267. Sandvik, T., Drew, T., Paton, D., 2000, CSF virus in East Anglia: where from? Vet Rec 147, 251. Terpstra, C., de Smit, A.J., 2000, The 1997/1998 epizootic of swine fever in the Netherlands: control strategies under a non-vaccination regimen. Vet Microbiol 77, 3-15. Vilcek, S., Herring, A.J., Herring, J.A., Nettleton, P.F., Lowings, J.P., Paton, D.J., 1994, Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch Virol 136, 309-323. Zhang, F., Yu, M., Weiland, E., Morrissy, C., Zhang, N., Westbury, H., Wang, L.F., 2006, Characterization of epitopes for neutralizing monoclonal A c c e p t e d M a n u s c r i p t -19 -antibodies to classical swine fever virus E2 and Erns using phagedisplayed random peptide library. Arch Virol 151, 37-54.

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Acknowledgements

We particularly thank Alison Ward, Nicola White and Mick Kirby for their excellent assistance with the animals. The efforts of Ian Wright, Meenakshi Khatri, Olubukola Sosan and JaeJo Kim, from the National Veterinary Research and Quarantine Service, Republic of Korea, in the processing of samples are also very much appreciated. This study was funded by DEFRA project SE0778.

A c c e p t e d M a n u s c r i p t -16 -which will allow prevention of shedding by vaccination to be assessed. The extended macro-and histopathological scoring systems described here will allow systematic comparison and assessment of pathological findings related to CSF infection.

Conflicts of interest

None