Department of Veterinary & Biomedical Sciences, University of Nebraska-Lincoln, Fair Street & East Campus Loop, Lincoln, NE 68583-0905, USA
Correspondence
Ruben Donis
rdonis{at}unlnotes.unl.edu
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ABSTRACT |
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INTRODUCTION |
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BVDV, BDV and CSFV are thought to have diverged from a common ancestor by adaptation to various species, including cattle, sheep and swine. Pestiviruses can cross host species barriers and infect other animal species in the order Artyodactyla. For example, ruminant pestiviruses often infect swine (Liess & Moennig, 1990). However, pestivirus infection in the non-adapted host (e.g. BVDV in swine) appears to be inefficient (Carlsson & Belak, 1994
). This scenario is in general recapitulated in vitro; pestiviruses show preference for cells of the species to which they are adapted: BVDV can replicate in swine cells, but only with greatly reduced efficiency (Flores et al., 1996
; Roehe & Edwards, 1994
).
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METHODS |
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Creation of chimeric BVDV genomes.
The chimeric BVDV genome with a substituted E2 glycoprotein gene was constructed by removing a segment of DNA encoding E2 in the BVDV infectious clone pNADLp15 (Vassilev et al., 1997) and inserting the homologous segment of BDV strain 31, giving rise to pN-E2bdv. To preserve E2 processing in the chimeras, the junctions were designed based on previously reported signalase processing sites at the termini of the bovine pestivirus E2 (Rumenapf et al., 1993
). A similar approach was followed to engineer a control chimeric virus, pN-890E2, expressing the E2 of BVDV strain 890, an isolate that belongs to genotype II (Fig. 2
) (Ridpath & Bolin, 1995
). The pN-E2bdv chimeric genome was used as a template for in vitro RNA synthesis (Megascript, Ambion). RNA transcripts were electroporated into bovine cells and virus replication was quantified by plaque assay, as described previously (Vassilev et al., 1997
).
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Normalized infection.
The input multiplicity was adjusted to obtain the same percentage of MDBK and LG-lamb cells infected with wt and chimeric virus. The percentage of infected cells in each monolayer was determined by indirect immunofluorescence on wells treated in an identical fashion. The medium and cells were harvested at 12 h p.i. by freeze-thawing and the total yield of infectious progeny was determined by plaque assay on LG-lamb cells.
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RESULTS |
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To evaluate the replication efficiency of chimeric BVDVE2bdv pestivirus in MDBK and ovine cells, we determined the yield of infectious progeny during a period of 24 h after infection (Fig. 3). The BVDVE2bdv virus produced tenfold less infectious virus progeny than the BVDV parent after 12 h of infection in MDBK cells. This difference in progeny yield increased to 100-fold at 24 h post-infection (p.i.). In contrast, the MDBK cells were equally permissive for BVDVE2bdv and BDV, due to the reduced fitness displayed by BVDVE2bdv in these cells, caused by the E2 substitution (Fig. 3
). E2 is thought to exist as a disulfide-linked heterodimer with E1 and to interact with Erns non-covalently. In addition, the E2 precursor E2P7 may interact with the core protein before the release of P7 (Elbers et al., 1996
). The failure of BVDVE2bdv to replicate in bovine cells could be an artefact stemming from the incompatibility of the BDV E2 with the other viral proteins. To address this question, we engineered the replacement of the BVDV E2 with the E2 from BVDV strain 890, which is classified as genotype II. We used this strain because it has the same level of E2 amino acid divergence from BVDV strain NADL as BDV strain 31. The amino acid sequences of BDV 31 and BVDV strain 890 E2 are both
58 % identical to that of strain NADL (GenBank accession nos AAB37578, AAA82981 and NP_044403, data not shown). The growth curves of BVDVE2gII in MDBK and ovine cells were very similar to that of the parental BVDV strain NADL (Fig. 3
). These results indicated that the E2 from a divergent pestivirus, such as a genotype II strain, can establish the putative functional interaction(s) with the rest of the genotype I BVDV proteins to replicate efficiently in cell culture (Elbers et al., 1996
). This finding is consistent with the hypothesis that the impaired growth of BVDVE2bdv in MDBK cells is not caused by defective interactions among viral proteins, but rather results from inefficient E2host interactions. Similarly, the efficient replication of BVDVE2bdv in ovine cells, found to be comparable with that of BDV, suggests that the ovine E2 can function efficiently with the other viral structural or non-structural proteins of BVDV that may interact with it.
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Taken together, these studies demonstrate that BDV E2 functions efficiently in the chimeric virus to infect cells derived from its natural sheep host but fails to mediate efficient infection of MDBK cells. E2 forms a heterodimer with E1, whose function is unknown (Weiland et al., 1990). The extensive sequence divergence between BDV and BVDV E2 is not accompanied by equally profound changes in E1 (Becher et al., 1999
; Deng & Brock, 1992
). However, our results suggest that the putative interactions of BVDV E1 with a heterologous E2 from BDV or BVDV type II remain functional in the chimeras.
The relative contribution of entry or assembly defects to the inefficient replication of BVDVE2bdv in MDBK as compared with LG-lamb cells cannot be derived from this study. Further research will be necessary to quantify the efficiency of each of these processes in BVDVE2bdv infections. These findings have potentially significant implications for the understanding of host factor involvement in the pestivirus replication cycle. Further mapping of the E2 domain that determines efficient infection of MDBK cells using a homologue-scanning strategy may shed light on BVDV assembly and infectivity in ruminant cell cultures (Morrison et al., 1994).
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ACKNOWLEDGEMENTS |
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Received 2 May 2002;
accepted 20 November 2002.