Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, Boddenblick 5a, 17493 Greifswald-Insel Riems, Germany
Correspondence
Martin Beer
beer{at}rie.bfav.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 5'-UTR, Npro, E2, NS3 and 3'-UTR sequences of isolate D32/00_HoBi reported in this paper are AY489116, AY489117, AY604725, AY713481 and AY604726, respectively.
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MAIN TEXT |
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D32/00_HoBi was isolated from fetal sheep thymus cells SFT-R [RIE43, Collection of Cell Lines in Veterinary Medicine (CCLV), Insel Riems] that were grown in Dulbecco's modified Eagle's medium supplemented with 10 % FCS batch 547 from Brazil (Biochrom). PCR analysis directly from the FCS sample, as well as inoculation of KOP-R cells, a diploid bovine oesophageal cell line (RIE244, CCLV), demonstrated clearly that this particular batch (547) was the source of contamination with D32/00_HoBi. Most efficient replication of the virus was observed with bovine cell lines. In contrast, no efficient virus propagation was detectable after inoculation of porcine kidney PK-15 cells (data not shown). D32/00_HoBi-infected KOP-R cells were tested in binding assays with a panel of pestivirus-specific mAbs directed against NS23, ERNS and E2 (Table 1; Peters et al., 1986
; Moennig et al., 1987
; Edwards et al., 1988
, 1991
; Paton et al., 1994
, 1995
). Immunofluorescence (IF) and immunoperoxidase analyses were performed as described previously (Depner et al., 2001
). It could be demonstrated that three of the five NS23-specific mAbs (C16, 103/105 and 435) were able to detect D32/00_HoBi-infected cells (Table 1
). Interestingly, the BVDV-1- and BDV-specific mAb 160 (Beer & Wolf, 1999
) was unable to bind, and only mAbs 103/105 and C16 reacted with all strains tested (Table 1
). In addition, only two (433 and 434) of the eight ERNS-specific mAbs tested, which normally detect BVDV-2 isolates, and none of the ten E2-specific mAbs reacted with D32/00_HoBi in the binding assay (Table 1
). This markedly restricted reaction pattern of the newly isolated virus was significantly different from those of other pestiviruses. However, failure of mAb 160 to bind to D32/00_HoBi-infected cells and interaction with the ERNS-specific mAbs 433 and 434 are related most closely to the reaction pattern of BVDV-2 and the Giraffe-1 strains (Table 1
).
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The RT-PCR products were cloned directly and three independent plasmid clones were sequenced by using M13 universal and reverse primers. Subsequently, a consensus sequence of the amplified and cloned fragments was generated. Part of the 5'-UTR (184 bp fragment; GenBank accession no. AY489116), the entire Npro sequence (504 bp; accession no. AY489117), the entire E2 sequence (1119 bp, accession no. AY604725), the entire NS3 sequence (2049 bp; accession no. AY713481) and part of the 3'-UTR (135 bp, accession no. AY604726) were aligned with the sequences of standard strains from GenBank (GCG programs; CLUSTALW; HUSAR, DKFZ Heidelberg), followed by phylogenetic analysis based on the Npro, E2 and NS3 alignments (Fig. 1ac). The trees were generated by using the PUZZLE software (Strimmer & von Haeseler, 1996
). For maximum-likelihood tree reconstructions, 1000 puzzling steps and the Hasegawa model for nucleotide substitution were used. Dendrograms generated by PUZZLE were visualized by using the TREEVIEW software (Page, 1996
). The Npro- and E2-based phylogenetic trees clearly showed six major branches corresponding to the four pestivirus species BVDV-1, BVDV-2, BDV and CSFV, the tentative species Pestivirus of giraffe and the novel strain D32/00_HoBi (Fig. 1a, b
). Phylogenetic analysis of NS3 revealed a clear distinction of all different species. However, due to the markedly higher identities of the NS3-encoding sequences between all pestivirus species, hypothetical taxonomical units were generated for BVDV types 1 and 2, as well as for the tentative species Pestivirus of giraffe and D32/00_HoBi (Fig. 1c
). The robustness of the obtained clades was supported by high probability values of 75100 % at the internal branch-points dividing the different species (Fig. 1a
c). A comparison of sequence identities was obtained with the program GAP (HUSAR, DKFZ Heidelberg). Based on the entire Npro-encoding sequence, none of the selected pestivirus species had >67 % identity to D32/00_HoBi (6467 %; Table 2
). Identity values for the entire E2 nucleotide sequence ranged between 58 and 62 %. In addition, analyses using the 5'-UTR and 3'-UTR sequences resulted in an identical genotype/species classification, with identities of 6675 % (5'-UTR) and 4155 % (3'-UTR) (data not shown). The highest identity values were calculated for the extremely conserved NS3 nucleotide sequences (7678 %). The calculated sequence identities within the Npro-encoding region compared with the other pestivirus species are similar to that reported for the giraffe strain (Avalos-Ramirez et al., 2001
), but 411 % lower than those between the different BDV genotypes (Table 2
).
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As D32/00_HoBi was isolated from an FCS sample, its real host remains unidentified. Nevertheless, domestic or free-ranging ruminants, particularly of the genus Bos within the family Bovidae, are the most likely target species of D32/00_HoBi. In a first animal experiment, pigs (n=2) and cattle (n=2) were infected intranasally with 2x108 TCID50 D32/00_HoBi per animal. No D32/00_HoBi virus could be reisolated from the inoculated pigs, none of the contact pigs seroconverted and no clinical symptoms or leukopenia could be detected. However, both inoculated pigs seroconverted versus D32/00_HoBi. In contrast, viraemia was detected at day 5 post-infection in the infected calves, D32/00_HoBi virus was shed for several days (days 36 post-infection) and slight leukopenia, as well as a mild increase in body temperature, could be detected (data not shown). As observed for the inoculated pigs, the infected calves did not show clinical signs during the whole experiment.
Taken together, comparative sequence analyses, binding studies with mAbs and cross-neutralization assays revealed marked differences between D32/00_HoBi and all current pestivirus species. We propose that the low nucleotide sequence identities between D32/00_HoBi and selected members of all other species based on the 5'-UTR, Npro, E2 and 3'-UTR sequences support classification as a novel pestivirus species; the low sequence similarities are consistent with previous values that have been used for division into species (Vilek et al., 1994
; Becher et al., 1997
, 2003
; Avalos-Ramirez et al., 2001
). Thus, D32/00_HoBi may be classified as a representative of a novel tentative species within the genus Pestivirus by analogy to the species Pestivirus of giraffe (van Regenmortel et al., 2000
). In addition, the NS3 nucleotide sequence comparison generated the same identity values between D32/00_HoBi and all other pestivirus species. Interestingly, the tree based on the NS3 sequences revealed a slightly enhanced phylogenetic relationship between the tentative species Pestivirus of giraffe and D32/00_HoBi, but also between both BVDV species. Therefore, a discussion about generally accepted criteria that are used for the classification of pestiviruses into species, genotypes and subtypes could be helpful, and D32/00_HoBi represents an important jigsaw piece.
Although isolated from a batch of FCS from Brazil, it has to be taken into consideration that D32/00_HoBi might be introduced into susceptible animals in Europe, e.g. via biological products containing FCS. It has previously been demonstrated that numerous animal cell lines contain BVDV (Bolin et al., 1994). Moreover, a Bovine herpesvirus 1 modified live vaccine was contaminated with virulent BVDV-2 (Falcone et al., 1999
). Assuring the absence of pestiviruses is therefore an important part of the quality control of biological products such as vaccines. For the detection of D32/00_HoBi in cell cultures, IF staining with NS3-specific pan-pestivirus mAbs (e.g. 103/105 and C16) can be used. In addition, RT-PCR could be used for testing sera, media or master stocks. However, due to the large sequence differences, established RT-PCR protocols might fail to detect D32/00_HoBi RNA. In this context, it must be mentioned that the well-known pan-pestivirus primer pair 324/326 (Vil
ek et al., 1994
), which can be used for the amplification of a fragment of approximately 290 bp in the 5'-UTR region, did not detect D32/00_HoBi (data not shown). As a consequence, diagnostic assays used for the determination of pestiviruses in biological products should be tested for their ability to detect D32/00_HoBi.
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ACKNOWLEDGEMENTS |
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Received 28 April 2004;
accepted 25 August 2004.
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