Institute for Experimental Pathology, University of Iceland, Keldur, IS-112, Reykjavík, Iceland1
Author for correspondence: Valgerdur Andrésdóttir. Fax +354 5673979. e-mail valand{at}hi.is
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Abstract |
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Introduction |
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Methods |
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Virus was propagated in monolayers of sheep choroid plexus cells (SCP) (Sigurdsson et al., 1960 ). Choroid plexus cells were grown at 37 °C in a humidified atmosphere of 5% CO2 in Dulbeccos modified Eagles medium (DMEM) supplemented with 200 units/ml penicillin and 100 units/ml streptomycin and either 10% or 1% lamb serum (for expansion of uninfected cells or for virus propagation, respectively). Virus isolation was by explantation and cocultivation.
Macrophage cultures were established as follows. Heparinized blood (100 ml) was collected from normal sheep, and peripheral blood mononuclear cells obtained by sedimentation on Histopaque-1077 (Sigma) were washed repeatedly in PBS and resuspended at a concentration of 12x106 cells/ml in growth medium supplemented with 5x10-5 M mercaptoethanol. After incubation at 37 °C in a humidified atmosphere of 5% CO2 for 24 h, supernatant and unattached cells were removed, and adherent cells were further incubated for at least 7 days before they were infected.
Infections were carried out at an m.o.i. of 0·20·5 TCID50. Each experiment was repeated at least two times. The replication of virus differed somewhat between macrophage preparations; however, differences between virus strains were very consistent within each experiment.
RT assay.
Viral particles from 0·5 ml of cell-free supernatants from infected cells were pelleted at 14000 r.p.m. in a microcentrifuge at 4 °C for 1 h. RT activity was determined as described previously (Andrésdóttir et al., 1998 ).
Neutralization tests.
Neutralization tests were carried out as published previously (Lutley et al., 1983). Briefly, 102 TCID50 of the virus in 0·1 ml was mixed with 0·1 ml of twofold dilutions of serum, mixtures were held at 4 °C for 48 h and then inoculated into four SCP cultures, which were observed for 23 weeks. Neutralization titres of the sera were calculated as the reciprocal of the serum dilution that caused complete neutralization in 50% of inoculated cultures.
Infection of cells and lysis for PCR.
SCP cells were infected with visna virus at an m.o.i. of 3 TCID50. When syncytia appeared (usually 23 days after infection), the monolayers of SCP cells were washed three times with PBS and the cells were suspended in 1 ml PBS using glass beads to remove the cells from the plastic. The cells were harvested and resuspended in 100 µl lysis buffer (10 mM TrisHCl, pH 8·0, 1 mM EDTA, 0·5% Triton X-100, 0·001% SDS, 300 µg/ml proteinase K) and incubated overnight at 37 °C. The enzyme was inactivated at 96 °C for 15 min. Three µl of the lysate were used for PCR amplification.
PCR, cloning and sequencing.
PCR was performed using DyNAzyme II DNA polymerase (Finnzymes Oy) and subjecting the samples to 30 cycles of 94 °C for 30 s, 53 °C for 30 s and 72 °C for 1 min. The PCR products were sequenced directly after purification of single strands using a biotin-labelled primer and streptavidin-coated magnetic beads, as specified by the manufacturer (Dynal). The following primer set was used for PCR amplification: 5' GAGGGATCAAGGATAAAAATGG 3' and 5' BioGGTATCGYTGCAGYAACAT 3'. Sequencing was performed using the primer 5' ATAGCACCATAACAGGAA 3'. The PCR products were also cut with XbaI and Sau3A and cloned into M13mp18 and M13mp19 for sequencing.
DyNAzyme, the DNA polymerase used in these studies, is one of the most accurate polymerases on the market, according to the producers (Finnzymes Oy). Our sequencing data support this statement, since we did not find any mutation in 5500 bases sequenced from PCR products of cloned DNA.
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Results |
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Using type-specific antiserum that neutralized the infecting strain K1514 but not a heterologous strain K796, it was found that ten (16%) of 61 virus isolates from blood escaped neutralization. These antigenic variants were isolated from six sheep and appeared randomly in time, except in one sheep (no. 1557) where the last four isolates from blood were variants. No correlation could be found between the replication of antigenic variants and disease progression (Lutley et al., 1983 ). Early appearance of neutralizing antibodies did, however, correlate with late onset of disease (Georgsson et al., 1993
).
Sequencing of a variable region in the env gene of the ten antigenic variants and 24 non-variants
We have mapped mutations changing a type-specific neutralization response in two molecular clones of visna virus to a highly variable region in the MVV outer glycoprotein (Skraban et al., 1999 ). We selected this region for sequencing in a number of virus isolates: the ten antigenic variants detailed above, and 24 isolates that were not antigenic variants. PCR was performed on virus isolated in SCP cells from experimentally infected sheep over the period 19731981 (Lutley et al., 1983) and fresh sheep samples were thus not available. Macrophages are considered to be the natural target cells of MVV, and the growth in SCP cells may select for SCP-tropic variants. However, we believe that the use of the SCP isolates is legitimate, since the infecting MVV strain, K1514, is permissive in SCP cells and we have shown that the cell tropism resides in the LTR (Agnarsdóttir et al., 2000
). The neutralization titres of a type-specific antiserum to the infecting strain K1514, against the virus isolates, are listed in Table 1
, as are the titres of a polyvalent antiserum obtained late in infection from a sheep infected with strain 1514 (serum taken at 72 months after infection from sheep 1521, see Fig. 2
). The antigenic variants that were not neutralized by the type-specific antiserum were all readily neutralized by the polyvalent serum.
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The time points of the virus isolations are listed in Fig. 1. Two of the antigenic variants were isolated 6 months post-infection, one after 15 months and the others later. The accumulation of mutations in the antigenic variants as opposed to non-variants cannot be explained by the antigenic variants being isolated at later time points, because nine of the non-variants were isolated 30 months post-infection or later.
Two clones of strain K1514 have been sequenced (Braun et al., 1987; Sonigo et al., 1985 ). The two clones diverge by nine nucleotides of 1965 in the SU of the env gene. Six of these mutations are non-synonymous and three are synonymous. We sequenced the entire genes for the SU of Env from three antigenic variants that were isolated from sheep 1557. We found 23 mutations that were not present in either of the two sequenced K1514 strains, counting mutations at each site only once. Four of these mutations were in the epitope region and all were non-synonymous. Of the remaining 19 mutations, ten (53%) were non-synonymous and nine (47%) were synonymous. The sequences have been submitted to GenBank.
Time of emergence of the antigenic variants with respect to neutralization profiles
Sera were drawn from all sheep at regular intervals and tested for neutralization of the infecting virus (K1514) and a heterologous visna virus, K796, which was not neutralized by type-specific antiserum against K1514 (Fig. 2). A type-specific neutralization response appeared in all sheep 25 months after infection. A more broadly reactive neutralizing response, which also neutralized strain K796 appeared much more irregularly. It appeared within a year in ten sheep and up to 4 years later in three others. Four additional sheep either developed low levels of neutralizing antibodies against K796 very late or did not develop any. In Fig. 2
, the time points of virus isolations are illustrated by arrowheads, and asterisks indicate neutralization escape mutants. Sixty-one blood isolates from 17 sheep were tested for neutralization with type-specific antiserum against K1514. Ten virus isolates from six sheep were not neutralized by the type-specific antiserum. Thirty-two virus strains were isolated at time points when there was no antibody against K796, and 29 were isolated in the presence of the broadly acting antibodies. All the escape mutants, except for one, were isolated at time points when only the type-specific antibodies were acting, before the emergence of the broadly reactive antibodies. One escape mutant was isolated just after the appearance of the broadly reactive antibodies when the neutralization titre for K796 had reached 8 (Fig. 2
, sheep 1521). The association between the appearance of the antigenic variants and the presence of type-specific neutralizing antibodies but absence of the broadly reacting antibodies was shown to be statistically significant using Fishers exact test (P=0·0135). The time function was not taken into account in this calculation. However, since the broadly reacting antibodies appeared later than the type-specific neutralizing antibodies, on the whole, virus isolated in the presence of the broadly reacting antibodies were isolated later and would therefore be expected to have accumulated more mutations.
Neutralizing antibody response following infection with a molecularly cloned virus
Three sheep (1951, 1954 and 1956) were infected intracerebrally with 0·4 ml of the molecularly cloned MVV KV1772kv72/67 with a titre of 107·3 TCID50/ml. Sera were collected every other week and tested for neutralization of two MVV strains: the infecting molecularly cloned strain, KV1772kv72/67, which has the same neutralization type-specificity as the strain K1554, and another molecularly cloned strain, LV1-1KS1, which is derived from strain K796 and is heterologous to KV1772kv72/67. All sheep had developed type-specific neutralizing antibodies by 12 weeks post-infection. The neutralization titre rose rapidly and was maintained throughout the 3 years that the sheep were kept alive. Antibodies that neutralized the heterologous strain LV1-1KS1 appeared later in two of the sheep, and not at all in one sheep (Fig. 3). Two virus isolates from each sheep were tested for neutralization by type-specific antiserum against the infecting strain, KV1772kv72/67, and a broadly reactive antiserum that neutralized both KV1772kv72/67 and the heterologous strain, LV1-1KS1. One isolate from sheep 1954 was not neutralized by the type-specific antiserum, and this antigenic variant arose in the absence of broadly reactive antibodies. All isolates were neutralized by the broadly reactive antiserum. The neutralization epitope region was sequenced in the six virus isolates. Eight mutations were found in this region in the antigenic variant, and these were distributed similarly to the mutations we had found in the other antigenic variants. There were mutations in the two linear epitopes, in one of the cysteines and in the potential glycosylation site. We sequenced two-thirds of the env gene in this variant and found it to be hypermutated compared with other virus isolates (data not shown). There were no mutations in the epitope region in the five isolates that were not antigenic variants (Fig. 4
).
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Replication rates of antigenic variants
To test the possibility that selection of the antigenic escape mutants could be explained by increased replication rates, we determined the rate of replication of two antigenic variants. The first was the antigenic variant that arose in the sheep infected with the molecularly cloned virus (isolate 1954-1636). Blood-derived macrophages were infected with the molecularly cloned virus KV1772kv72/67 or the antigenic variant 1954-1636 at an m.o.i. of 0·2. Virus replication was monitored by measuring RT activity daily in the cell supernatants. The replication rate of the antigenic variant was considerably slower than that of the parent virus, KV1772kv72/67 (Fig. 5). The other antigenic variant that was tested was isolate 165 from sheep 1521 (see Fig. 2
). This antigenic variant had a Cys
Tyr mutation (Fig 1
) and could be expected to have a disrupted three-dimensional structure, possibly affecting replication proficiency. Cultures of blood-derived macrophages were inoculated with four virus isolates from this sheep: an isolate taken at 2 months after infection (isolate 1521-49), 15 months (isolate 1521-165, the antigenic variant), 30 months (isolate 1521-256) and 60 months (isolate 1521-522). The antigenic variant (isolate 1521-165) grew distinctly slower than the other virus isolates (Fig. 6
). Both antigenic variants (1954-1636 and 1521-165) had emerged in the presence of type-specific neutralizing antibodies, despite the fact that they were impaired in growth.
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Discussion |
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In an MVV infection, type-specific neutralizing antibodies appear 25 months after infection, and in most sheep, other more broadly neutralizing antibodies appear up to 4 years later. The broadening of the neutralizing antibody response has been suggested to be due to mutants arising in the virus population that were antigenically related to the heterologous test strain (Pétursson et al., 1976 ). The low level of heterogeneity in the env genes of the virus isolates that were isolated in the presence of broadly reactive antibodies suggests, however, that the development of broadly reactive antibodies is more likely due to a reaction to conserved epitopes that are less immunogenic. The finding that all the antigenic variants were neutralized by the broadly active serum is further evidence for the conserved nature of these epitopes. Similar results have been obtained in studies of SIV (Clements et al., 1995
) and HIV-1 (Zhang et al., 1999
).
The role of humoral immunity in lentiviral infections is still a matter of controversy. It has been disputed whether antigenic variants of lentiviruses arise as a result of immune selection. Although antigenic variants can be selected in the presence of antibodies in vitro (Dubois-Dalq et al., 1979 ; Yoshida et al., 1997
), the functional role of antibodies under in vivo conditions is uncertain. It has been proposed that since neutralizing epitopes are located in regions that tolerate high variability and because of the high mutation rate of the lentiviruses, mutants with a selective disadvantage will be selected against, resulting in enrichment of variants with mutations in the neutralizing epitopes. These may then acquire dominance in the virus population, either accompanying another mutation, resulting in a more fit variant, or by bottleneck transmission (Domingo et al., 1993
). It is also possible that a change in the neutralization epitope coincides with a change in a recognition site for a receptor or coreceptor, as has been found in HIV (McKnight et al., 1995
). The region of the env gene that was compared in this study has indeed been shown to be highly variable, both between distantly related MVVs (Sargan et al., 1991
) and the more closely related Icelandic maedi and visna viruses (Andrésdóttir et al., 1998
). This region is within the fourth variable domain (V4) of MVV, which is analogous to V4 identified in the caprine arthritisencephalitis virus surface glycoprotein (Valas et al., 2000
). The higher proportion of non-synonymous nucleotide substitutions in this region than in other regions of the Env protein is suggestive of positive selection in this region. However, since we sequenced PCR products derived from virus propagated in SCP cells, we cannot rule out the possibility that growth in cell culture could have selected some of the mutations. The finding that the antigenic variants emerged at time points when only the type-specific antibodies were acting strongly suggests, however, that the type-specific antibodies provide the selective force. The correlation of early appearance of neutralizing antibodies with late onset of disease (Georgsson et al., 1993
) is further evidence that the antibodies are effective in limiting the spread of virus in the body.
Two clones of MVV strain K1514 used as inoculum in this study have been sequenced. The two genes of gp135 have been found to differ by nine mutations or 0·5% (Braun et al., 1987 ; Sonigo et al., 1985
). The sheep were inoculated with 106107 TCID50 of virus. It is therefore likely that some of the mutations resulting in antigenic variation may have been present in the inoculum, whereas others have evolved in the sheep.
It appears that there is enrichment of virus variants that escape neutralization in the presence of type-specific neutralizing antibodies. When the broadly neutralizing antibodies appear, the escape variants have no advantage, and some of them may be impaired in replication, and therefore they are not isolated as frequently. The escape mutants that we tested for growth rate in this study did in fact grow more slowly than the parent strains, while the growth rate of other antigenic variants may not be affected (Skraban et al., 1999 ). The humoral immune response therefore seems to be effective at limiting the spread of virus in an MVV infection, but the fact remains that there is no evidence for involvement of the antigenic variants in progression of the disease.
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Acknowledgments |
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Footnotes |
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b Present address: deCode Genetics, Reykjavík, Iceland.
c Present address: SigmaAldrich Fine Chemicals, St Louis, MO 63103, USA.
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References |
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Received 7 February 2002;
accepted 5 June 2002.