During 2004–2007, serum samples from 1,036 sheep and goats in Turkey were serologically screened for infection with pestiviruses of small ruminants. Of these, 11 serum samples from 7 sheep herds gave positive or doubtful reactions in the CSFV antibody–specific ELISA (HerdChek, IDEXX) and were subjected to commonly used virus neutralization testing (VNT) (7). VNT against the 2 established CSFV strains Alfort187 (genotype 1.1) and Diepholz (genotype 2.3) and against the BDV strains Moredun (genotype 1) and Gifhorn (genotype 3) revealed higher BDV titers in only 3 serum samples (Table 1). Equal or slightly higher titers against the CSFV reference strains became evident in 8 of the 11 serum samples, which came from 5 regions of Turkey. Further VNT analyses with the 2 previously obtained isolates, Aydin/04 and Burdur/05, demonstrated neutralizing antibody titers that were equal or higher than those against BDV and CSFV test strains. To elucidate the reason for strong serologic reactivity in CSFV assays, we genetically and antigenically characterized pestiviruses Aydin/04 and Burdur/05.

The complete genome sequence of Aydin/04 was determined as reported previously (8). The genome sequence of Burdur/05 was determined by next-generation sequencing on an Illumina MiSeq platform (2 × 250-bp paired end run, 593,328 reads) as recently described (9). Template total cellular RNA was extracted from supernatant of sheep fetal thymus cells. Of all reads, 73.9% were found to be of host origin. Of the nonhost reads, 89.9% assembled into a single sequence contig encompassing the entire pestivirus Burdur/05 genome (coverage 196-fold).

Figure 1

Figure 1. Phylogenetic and antigenic tree displaying relatedness of pestiviruses Aydin/04 and Burdur/05 to other Pestivirus species. A) For phylogenetic analysis, deduced polyprotein sequences from GenBank were used (CSFV: J04358, GU233734, JX218094, AY568569,...

Sequence and phylogenetic analyses were performed with complete genome sequences and deduced amino acid sequences of new pestiviruses Aydin/04 (GenBank accession no. JX428945) and Burdur/05 (KM408491). For further analyses, reference sequences were obtained from GenBank (Figure 1). Genetic distances were calculated by using the Kimura 2-parameter substitution model, and phylogenetic analyses were conducted by applying the neighbor-joining method as commonly used for CSFV phylogeny (11). With the same set of sequences, a grouping scan was performed by using the SSE platform (12). Comparison of the complete coding sequences of Aydin/04 and Burdur/05 revealed a genetic distance of 16.5%. Phylogenetic analyses based on deduced polyprotein sequences showed that isolates Aydin/04 and Burdur/05 form a distinct group located between CSFV and BDV (Figure 1, panel A).

Systematic antigenic characterization was performed by using cross-neutralization assays (Table 2). For this purpose, CSFV and BDV reference strains for which homologous serum was available were tested by VNT as described (7). In general, neutralization of both isolates was more efficient when performed with different CSFV antiserum than with BDV antiserum. In addition, the Aydin-specific antiserum obtained from animal experiments neutralized the CSFV reference strains with titers higher than those for the BDV strains (Table 2). Because no experimental infection with Burdur/05 has been performed, Burdur/05-specific antiserum was not available; however, close antigenic relatedness of both isolates was demonstrated by the high neutralization titers of the Aydin-specific antiserum for isolate Burdur/05 (Table 2). To quantify and to depict the antigenic relatedness, we calculated coefficients of antigenic similarity (R values) as described previously (13). An antigenic tree graphically displaying the R values clearly shows 2 distinct clades, one representing CSFV and the other comprising BDV strains (Figure 1, panel B). Furthermore, Aydin/04 is antigenically more closely related to CSFV than to BDV, but it also clearly differs from these 2 pestivirus species.

Because of their close relationship to CSFV, it was of particular interest to determine the ability of these ruminant pestiviruses to infect pigs and induce clinical disease. Therefore, 3 clinically healthy and pestivirus uninfected weaner (6 weeks of age) piglets were inoculated with 1 × 10⁶ 50% tissue culture infectious doses of isolate Aydin/04 and given a booster of 3 × 10⁷ 50% tissue culture infectious doses 2 weeks later. Pigs showed no clinical signs of disease, no fever, no platelet or leukocyte depletion, and no viremia (data not shown). For all 3 animals, strong seroconversion was found (50% neutralizing titer of serum for homologus virus was 240–640 on postinoculation day 77).

Figure 2

Figure 2. Amino acid similarity of pestiviruses Aydin/04 and Burdur/05 to representative CSFV and BDV polyprotein sequences. The same CSFV and BDV polyprotein sequences as in Figure 1 were used...

Several new genetically diverse groups of pestiviruses have emerged in domestic livestock and wild animals, adding to the continuously growing list of approved and tentative pestivirus species (1). According to phylogenetic analyses of short partial genome sequences, 2 pestivirus isolates, Aydin/04 and Burdur/05, recently circulating in sheep and goat herds in different regions of Turkey, were classified as novel members of the BDV species (6). However, the data from this study demonstrate that these novel Aydin-like pestiviruses are representatives of a new pestivirus species, genetically and antigenically located between CSFV and BDV (Figure 1). The genetic distance of 16.5% between these isolates indicates that distinct ruminant Aydin-like pestiviruses circulate in different regions of Turkey. For some genomic regions, both ruminant pestivirus isolates display an even higher similarity to CSFV than to BDV (Figure 2). The close genetic relatedness to CSFV is in line with the antigenic characterization by cross-neutralization assays as depicted in the antigenic tree (Figure 1, panel B). This finding is in contrast with findings for pestivirus isolates from Tunisia, another group of ruminant pestiviruses genetically closely related to CSFV but antigenically more closely related to BDV (14). The close antigenic relationship to CSFV explains the observed strong cross-reactivity of serum from sheep and goat in CSFV-specific ELISAs, even when the variable E2 protein is used as diagnostic antigen (Table 1). In routine diagnosis, questionable ELISA results are further investigated by VNT against CSFV and other pestiviruses (e.g., BVDV and BDV). Usually, VNT titers are highest for the homologous pestivirus species. Remarkably, even if representatives of the Aydin-like pestiviruses were included as test strains in the VNT, CSFV infection still could not be ruled out.

Although these novel pestiviruses are the closest known relatives of CSFV, experimental infection of pigs with Aydin/04 did not result in detectable viremia and clinical signs. Nevertheless, these ruminant pestiviruses are candidates for a switch to porcine hosts after ongoing virus evolution, which would have severe consequences for serologic diagnosis of classical swine fever, affecting control and monitoring programs performed in many parts of the world.