Bundesforschungsanstalt für Viruskrankheiten der Tiere, Paul-Ehrlich-Str. 28, D-72076 Tübingen, Germany
Correspondence
Gregor Meyers
gregor.meyers{at}tue.bfav.de
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ABSTRACT |
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Present address: Boehringer Ingelheim Vetmedica GmbH, D-55216 Ingelheim am Rhein, Germany.
Present address: Klinische Immunologie, Veterinärmedizinische Universität Wien, Veterinärplatz 1, A-1210 Vienna, Austria.
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INTRODUCTION |
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The causative agent of CSF, classical swine fever virus (CSFV), is classified as a species of the genus Pestivirus within the family Flaviviridae. Three other pestivirus species are known that are predominantly found in ruminants: two types of bovine viral diarrhoea virus (BVDV-1 and BVDV-2) and border disease virus of sheep (Heinz et al., 2000).
Like other members of the family Flaviviridae, pestiviruses are small, enveloped viruses with a single-stranded RNA genome of positive polarity. The pestivirus RNA lacks both 5' cap and 3' poly(A) sequences, and contains one long open reading frame encoding a polyprotein of about 4000 amino acids, which encompasses all virus proteins arranged in the order NH2NproCErnsE1E2p7NS2NS3NS4ANS4BNS5ANS5BCOOH. The polyprotein gives rise to 11 or 12 final cleavage products by co- and post-translational processing involving cellular and virus proteases (Lindenbach & Rice, 2001). Protein C and the glycoproteins Erns, E1 and E2 represent structural components of the pestivirus virion (Thiel et al., 1991
). E2 and, to a lesser extent, Erns are targets for antibody neutralization (Donis et al., 1988
; Weiland et al., 1990
, 1992
; Paton et al., 1992
; van Rijn et al., 1993
). Erns lacks a typical membrane anchor and is secreted in considerable amounts from infected cells. A highly unusual feature of this protein is its RNase activity, which was first identified by characteristic sequence motifs and then proven by enzymic tests with the purified protein (Schneider et al., 1993
; Hulst et al., 1994
; Windisch et al., 1996
). The function of this enzymic activity in the virus life cycle is presently unknown. Experimental destruction of the RNase by site-directed mutagenesis resulted in a virus that has growth characteristics in cell culture equivalent to those of wild-type (wt) virus (Hulst et al., 1998
; Meyers et al., 1999
; Meyer et al., 2002
). In animal experiments RNase-negative mutants of CSFV were found to be attenuated. The degree of attenuation varied for different mutations from completely apathogenic to low pathogenic, characterized by somewhat milder disease signs that vanished rapidly around day 10 post-infection (p.i.) (Meyers et al., 1999
). Similarly, a highly pathogenic BVDV-2 isolate was considerably attenuated by an RNase mutation that also resulted in rapid recovery of the animals and clearance of the virus (Meyer et al., 2002
). These results support a model in which the RNase activity of Erns interferes with the immune response of the animal host. However, the mechanisms of immunosuppressive activity remain unclear. A completely apathogenic CSFV RNase mutant was shown to be unable to produce the B-cell depletion characteristic for CSFV infection in pigs. This finding suggests a role for RNase in the process of B-cell reduction. We describe here the results of animal experiments conducted with mildly pathogenic RNase mutants of CSFV to analyse their effects on different populations of peripheral blood leukocytes.
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METHODS |
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Infection of cells and immunofluorescence assay.
As pestiviruses tend to be associated with host cells, lysates of infected cells were used for infection of culture cells. Lysates were prepared by freezing and thawing cells 48 h post-infection, and were stored at 70 °C. If not indicated differently in the text, an m.o.i. of about 0·01 was used for infection of cells.
For detection of infected cells in immunofluorescence assays, cells were fixed with ice-cold methanol/acetone (1 : 1) for 15 min, air-dried, rehydrated with PBS, then incubated with anti-CSFV mAb a18 which detects the E2 protein (Weiland et al., 1990). Bound antibodies were detected with an FITC-conjugated goat anti-mouse serum (Dianova).
Generation of mutant C-W300G.
Restriction, cloning and other standard procedures were carried out essentially as described previously (Sambrook & Russell, 2001). Restriction and modifying enzymes were obtained from New England BioLabs, Amersham, Invitrogen and Roche.
Starting with plasmid p666 (Meyers et al., 1999), single-strand-based mutagenesis was performed according to the method of Kunkel et al. (1987)
using Escherichia coli CJ236 cells (Bio-Rad), the VCMS single-strand phage (Stratagene) and oligonucleotide Ol-W300G (5'-TTACATGGGATCGGGCCCGAGAAA). A cDNA fragment containing the desired mutation and no second-site changes was obtained by cleavage with XhoI and SdaI and inserted in the full-length cDNA clone pA/CSFV (Meyers et al., 1996
), restricted with the same enzymes. The full-length plasmid with the desired mutation was linearized by SrfI cleavage and served as a template for transcription of a cRNA that was used for transfection of SK6 cells, as described previously (Meyers et al., 1996
).
RT-PCR.
RT-PCR was carried out with about 2 µg total cellular RNA isolated from infected cells by the Trifast method according to the supplier's protocol (Peqlab). The RT-PCR was done with the One-Step RT-PCR kit as recommended (Qiagen). The following PCR primers were used. Upstream: 5'-CATGCCATGGCCCTGTTGGCTTGGGCGGTGATA (positions 1033 of the primer correspond to nucleotides 11201143 of the CSFV Alfort/Tübingen genome); downstream: 5'-GGAATTCTCAGGCATAGGCACCAAACCAGG (positions 1130 of the primer correspond to nucleotides 18351854 of the CSFV Alfort/Tübingen genome).
Amplified cDNA fragments were purified by preparative agarose gel electrophoresis. Elution of DNA from the agarose gel was done with a Nucleotrap kit (Macherey Nagel). For sequencing with the upstream primer, the Big Dye Terminator Cycle Sequencing kit (Perkin Elmer Applied Biosystems) was used. Analysis of the sequencing products was done with an ABI Prism 377 DNA sequencer (Perkin Elmer Applied Biosystems).
Determination of RNase activity.
Determination of RNase activity was carried out essentially as described before (Schneider et al., 1993; Meyers et al., 1999
). If not specified, assays were conducted in a total volume of 200 µl containing 5 or 50 µl supernatant of the second centrifugation step (60 min at 4 °C and 45 000 r.p.m., TLA 45 rotor, Beckman tabletop ultracentrifuge TL100) and 80 µg poly(rU) (Pharmacia) in RNase assay buffer (40 mM Tris/acetate, pH 6·5, 0·5 mM EDTA, 5 mM dithiothreitol). After incubation of the reaction mixture at 37 °C for 1 h, 200 µl 1·2 M perchloric acid and 20 mM lanthanum sulfate was added. After 15 min incubation on ice, the mixture was centrifuged for 15 min at 4 °C and 14 000 r.p.m. in an Eppendorf centrifuge. Three volumes of water were added to the supernatant and an aliquot of the mixture was analysed by measuring the absorbance at 260 nm using an Ultrospec 3000 spectrophotometer (Pharmacia). As a positive control, RNase A from bovine pancreas (Serva) with an activity of 85 Kunitz units (mg protein)1 was used instead of the cell extract.
Animal experiments.
For each CSFV variant, three or four piglets (German Landrace; 2025 kg) were used. If not specified, the infection dose was 0·5x105 to 1x105 TCID50 per animal, depending on the size of the animals; two-thirds of the inoculum was administered intranasally (one-third in each nostril), one-third intramuscularly. The different groups were housed in separate isolation units. Blood was taken from the vena jugularis at the time points indicated. Coagulation was inhibited with heparin [20 IU (ml blood)1] or sodium citrate (3·8 % w/v).
To test the animals for the presence of virus in the blood, SK6 cells seeded in a 24-well plate were incubated with 106 isolated peripheral blood leukocytes (prepared as described by Saalmüller et al., 1987) and 150 µl medium. After 1 h at 37 °C, the mixture was removed and the cells were washed twice with medium and incubated for 4872 h at 37 °C. Infection of cells was demonstrated by immunofluorescence.
Isolation of peripheral blood mononuclear leukocytes (PBMC) and flow cytometric analyses (FCM).
PBMC were isolated from heparinized blood samples by Ficoll-Hypaque (Pharmacia) centrifugation (800 g, 30 min) as described previously (Saalmüller et al., 1987).
Leukocytes were stained for two-colour FCM for detection of B-lymphocytes, monocytes and granulocytes in a three-step procedure, as follows. (i) Incubation of a mixture of 100 µl PBS and 100 µl heparinized blood with mAb B-ly4 (anti-human CD21, IgG1; Pharmingen) and mAb 74-22-15A [anti-SWC3, IgG2b; J. K. Lunney, USDA ARS, Beltsville, MD, USA (Pescovitz et al., 1984)] for 20 min on ice. (ii) Fixation of leukocytes and lysis of erythrocytes by adding 100 µl Novalyse (Dianova) for 15 min at room temperature and addition of 1 ml water for an additional 5 min; thereafter cells were washed twice with PBS2 % v/v FCS. (iii) Incubation with isotype-specific conjugates (anti-IgG2b-FITC, labelled with FITC; anti-IgG1-PE, labelled with phycoerythrin; Southern Biotechnology Associates). T-lymphocyte subpopulations (Saalmüller et al., 1987
, 1999
) were analysed by labelling with mAbs against the leukocyte-differentiation antigens CD4 and CD8: mAb 74-12-4 (IgG2b; Pescovitz et al., 1985
) and mAb 11/295/33 (IgG2a; Jonjic & Koszinowski, 1984
), respectively. Binding of specific antibodies was visualized with isotype-specific conjugates (anti-IgG2b-FITC and anti-IgG2a-PE) as above. For the detection of
-T-lymphocytes (Saalmüller et al., 1990
) and the classification of their CD8-defined phenotypes, a combination of mAb PPT16, directed against the porcine TcR-
chain (IgG2b, a gift from H. Yang and R. M. Parkhouse, IAH Pirbright Laboratory, Pirbright, UK; Davis et al., 1996
) and CD8 (11/295/33, IgG2a) together with the respective isotype-specific conjugates (Southern Biotechnology Associates) was used. Natural killer cells with the phenotype CD2+ CD5 (Saalmüller et al., 1994
) were detected by labelling the leukocyte population with a combination of mAbs directed against CD2 (MSA4, IgG2a; Hammerberg & Schurig, 1986
) and CD5 (b53b7, IgG1; Saalmüller et al., 1994
) together with the isotype-specific conjugates anti-IgG1-PE and anti-IgG2
-FITC (Southern Biotechnology Associates) in a next step. The presence of T-helper memory cells (Saalmüller et al., 2002
) in the leukocyte population was monitored by an antibody combination against CD4 (74-12-4, IgG2b) and MHC class II (MSA3, IgG2a; Hammerberg & Schurig, 1986
) and the respective isotype-specific conjugates.
Between each of the incubation steps, cells were washed with PBS2 % FCS. All analyses were performed on an FACStarplus (Becton Dickinson) as described previously (Saalmüller et al., 1994).
Percentages of positive cells were calculated using the software WINmdi (freeware by joe trotter) or CELL QUEST (Becton Dickinson). For discrimination between SWC3-positive monocytes and granulocytes, electronic gates in the forward-scatter (FSC) vs side-scatter (SSC) plot were used. For calculation of lymphocyte subsets, only cells in the electronic-based lymphoid gate of FSC vs SSC were analysed.
The absolute number of leukocytes was determined by testing the blood of the respective animals in a Coulter counter.
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RESULTS |
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The results of the experiment conducted with C-H297K were comparable with published data (Meyers et al., 1999): viraemia was detected for 58 days and the pigs developed fever (Fig. 1
) and mild disease signs but then recovered fully between days 10 and 13 p.i. The control animals infected with wt CSFV showed a similar course of disease in the initial phase, but exhibited severe clinical signs comprising diarrhoea, anorexia, ataxia, severe weakness and respiratory problems later on. Hyperthermia started at day 4 p.i. and persisted until the animals were put down in a moribund state at day 10 or 12 p.i. (animals #15/6 and #15/7, or #15/8 and #15/9, respectively). The wt CSFV-infected animals were viraemic from day 3 p.i. until death, whereas pigs infected with the mutant contained the virus only transiently in the blood (days 37, or 10 p.i.).
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Analysis of the white blood cells showed a clear leukopenia for all the animals included in the two studies (Fig. 1). In contrast to the experiment with mutant C-H346
, the mutant that did not induce any clinical signs in previous experiments, we observed a significant decrease in B-cell numbers, not only for the animals infected with wt CSFV, but also for the two different mutants (Fig. 1
). However, in comparison with the wt virus-infected pigs, the level of leukocytes and B cells started to increase again after 6 or 7 and 9 or 10 days p. i., respectively. For the reasons outlined above, animal #7/1 needed longer to restore leukocyte numbers. Taken together, the two RNase mutants tested here were different from the completely attenuated mutant C-H346
, as they induced a clearly detectable reduction of B-cell numbers in a range comparable to wt CSFV. The recovery of cell numbers clearly correlates with the general recovery of the animals and can hardly be regarded as a primary effect of the destruction of the RNase.
Infection with RNase-negative or wt CSFV has equivalent effects on the composition of peripheral blood cells in pigs
To find out whether there is a difference between animals infected with RNase-negative and wt virus with regard to other leukocyte populations in the peripheral blood, a further animal experiment was conducted with mutant C-W300G and a variety of different parameters were tested. The disease signs were similar to those observed in the previous experiments. For the animals infected with the mutant, mild signs of disease, elevation of body temperature, leukopenia and reduction of B-cell numbers were observed during the first approximately 10 days p.i. followed by a sudden recovery (Fig. 2a). Blood cell viraemia was detected for up to 5 days (days 59 p.i.). Controls infected with the wt virus developed fatal CSF with continuous pyrexia, viraemia (from day 2 p.i. until death), anorexia, diarrhoea, ataxia, respiratory problems, leukopenia and B-cell depletion (Fig. 2a
), and had to be put down on day 12 or 13 p.i. in a moribund state of health. In addition to leukocyte and B-cell numbers, the numbers of natural killer cells, granulocytes, monocytes, TcR-
T cells, cytolytic T cells and T-helper cells in the peripheral blood were also determined. The latter fraction was further characterized with regard to the content of naive MHC class-II-negative versus MHC class-II-positive activated or memory cells (Fig. 2b
; data not shown). No significant difference between animals infected with wt CSFV or C-W300G was found for any of the cell types tested. However, the attenuated nature of C-W300G was obvious from the recovery of cell numbers starting around days 1012 p.i. A further parameter demonstrating the attenuation of RNase-negative mutants was observed when we determined the degree of virus infection for some of the cell types by detection of the CSFV E2 protein. Considerable numbers of virus-positive cells were detected only in animals infected with wt CSFV, with very high levels in granulocytes and monocytes (Fig. 2b
). Similarly to the findings reported for C-W300G, an equivalent experiment with the attenuated mutant C-H297K did not lead to detection of virus in the different leukocyte populations in the FACS analysis (data not shown). Thus, the degree of infection of blood leukocytes appears to be a good parameter by which to judge the virulence of CSFV in the natural host.
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DISCUSSION |
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CSFV is known to induce a specific reduction in B-cell numbers in the blood and different organs of the infected pig (Susa et al., 1992). This reduction exceeds by far the general leukopenia observed during CSF in pigs, and represents an early marker of CSFV infection. It was therefore attractive to speculate about a connection between RNase activity of Erns and destruction of B cells. The first analyses conducted with one RNase-negative CSFV mutant (C-H346
) showed that the reduction in B-cell numbers was not detectable in animals infected with the mutant (Meyers et al., 1999
). However, C-H346
was exceptional as it was completely apathogenic and did not result in substantial viraemia, whereas other RNase-negative CSFV variants induced clearly detectable disease signs and could consistently be recovered from the blood of infected pigs. It is therefore apparent that the phenotype of C-H346
is not only due to lack of the RNase activity, but results from the destruction of the RNase and an additional factor that is not further defined. We therefore repeated the analysis with other RNase-negative CSFV mutants and revealed that the absence of B-cell depletion is not a general feature of animals infected with such Erns mutants. This result parallels experiments with equivalent BVDV mutants in cattle (Meyer et al., 2002
). B-cell depletion is not observed in BVDV-infected cattle, and comparison of the B-cell numbers in the peripheral blood of animals infected with wt BVDV or RNase-negative BVDV mutants did not reveal any differences. Taken together, the data obtained for the different CSFV and BVDV mutants indicate that B-cell depletion is not induced by the Erns RNase, and it seems likely that B cells do not represent the target of the RNase.
Based on the finding that destruction of the RNase activity of Erns leads to virus attenuation and on the putative cytotoxic potential of the enzyme, we searched for effects on the composition of peripheral blood cells in infected pigs that could be due to the activity of the RNase. The general leukopenia induced by the two types of virus showed equivalent levels. We compared the changes induced by infection with RNase-negative or wt CSFV on granulocytes, monocytes, different T-lymphocyte subpopulations (TcR- T cells, cytotoxic T cells, total T-helper cells, naive T-helper cells, activated and memory T-helper cells) and natural killer cells (part of the data not shown). Reduction was detected for all leukocyte subpopulations, but no significant differences between animals infected with RNase-negative or wt CSFV were found during the initial phase of infection. Later, the numbers of all tested cells recovered in the animals inoculated with RNase mutants, but this effect coincides with the general recovery of the animals. It can therefore be concluded either that the RNase does not affect the peripheral blood cells in a way that changes the overall leukocyte composition or that these changes are not prominent enough to be detected in our assays. The most obvious difference observed during our analyses concerned the virus load in the blood of infected animals, which differed dramatically between wt virus and mutants. This result can certainly be regarded as further proof of the attenuation of RNase-negative viruses, and explains why wt CSFV-infected animals develop more severe disease signs. However, this difference cannot answer questions concerning the target of the RNase and the mechanism by which it increases virus virulence. Further analyses are necessary that will need to include further cell types, for example bone marrow cells, which have been shown to undergo apoptosis in response to CSFV infection in tissue culture experiments (Summerfield et al., 2000
, 2001b
).
The mutant C-W300G was found to revert to the wt virus in pigs soon after infection. We were not able to isolate the mutant virus from the infected animals. Even virus obtained at the earliest viraemic time-points apparently did not represent a mixture, but displayed an unambiguous sequence with TGG at codon 300. Similarly, only the revertant could be isolated from nasal swabs. Thus C-W300G virus was not secreted from the infected cells at the site of infection (two-thirds of the inoculum administered into the nose). By contrast, the mutant was stable during propagation in tissue culture in a porcine spleen cell line (SK6) and a pig lymphoma cell line (38A1D). However, similarly to the situation within the natural host, reversion occurred in STE and MAX cells. This finding cannot be explained by a tendency to restore RNase activity, as we never found any indication of reversion in a variety of other RNase-negative viruses, although the H297L and H346L mutants would also be able to revert by single base substitutions. It therefore has to be concluded that the revertants are selected by certain cell types because of an advantage concerning another feature of Erns, for example its function during infection of cells. The rapid reversion within the animal shows that the wt virus has to have a significant advantage over the mutant for propagation in the tissues or cells representing the primary replication sites of the virus. As little is known about the function of Erns as a structural protein and its possible interaction partner(s) on the surface of the host cell, discussion of the molecular basis of this finding would be premature. A more urgent question is why C-W300G is obviously attenuated although there is prompt reversion to wt virus. CSFV live vaccines are known to induce protective immunity quickly. It therefore might be that the time between infection and the presence of significant amounts of revertants is sufficient to prime the immune system in a way that allows the pig to mount a timely immune response. Alternatively, the effect of the Erns RNase might be an early event so that a certain target has to be hit once in the initial phase of the disease to interfere with the immune response. Further experiments are necessary in order to distinguish between these possibilities. It will be very interesting to elucidate the mechanism behind the obvious attenuation of RNase-negative pestiviruses, but the present report indicates that this will require alternative approaches.
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ACKNOWLEDGEMENTS |
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Received 20 January 2004;
accepted 26 March 2004.
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