Antigenic Drift in Visna: Virus Variation During Long-term Infection of Icelandic Sheep

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1 J. gen. Virol. (1983), 64, Printed in Great Britain 1433 Key words: visna virus~antigenic drift/persistent infection/retroviruses Antigenic Drift in Visna: Virus Variation During Long-term Infection of Icelandic Sheep By ROGER LUTLEY, GUDMUNDUR PI~TURSSON, PALL A. PALSSON, GUDMUNDUR GEORGSSON, JOHN KLEIN I AND NEAL NATHANSON 1. Institute for Experimental Pathology, University of lceland, Reykjavik, Iceland and 1Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. (Accepted 24 February 1983) SUMMARY A group of 20 Icelandic sheep were infected intracerebrally with visna virus strain 1514, and 209 virus isolates were obtained from the blood, cerebrospinal fluid, and central nervous system (CNS) over a period of 7 years, during which eight animals developed clinical signs of visna necessitating sacrifice. (i) Using type-specific antisera, it was found that 12 (16%) of 76 isolates tested escaped neutralization. These 12 variant viruses were distributed randomly among animals and over time, and did not replace the infecting strain even though all sheep developed homotypic antibody within 3 months of infection. The one exception was sheep no (an animal without clinical visna), where the last six isolates were variants. (ii) A total of 35 blood and CNS isolates from seven of these sheep (including five with clinical visna) were tested against serial samples of their own sera. Autologous antisera neutralized all isolates tested with the exception of isolates from sheep None of the isolates obtained at sacrifice from the five sheep with clinical visna escaped neutralization with autologous antisera. These data suggest that although variant viruses are encountered at considerable frequency during long-term infection of Icelandic sheep, the variants usually do not replace the infecting strain. Antigenic drift does not appear to be essential for virus persistence or for the development of clinically evident CNS lesions. INTRODUCTION Visna is a chronic central nervous system (CNS) infection of sheep with a naturally occurring ovine retrovirus (Haase, 1975 ; P&ursson et al., 1976,!978, 1979). The virus persists indefinitely after infection, although at low levels, and the infected sheep develop subacute encephalitis which may progress to clinical paralysis at irregular intervals from a few months to many years after inoculation. Infected Icelandic sheep regularly develop neutralizing antibodies in their serum within 3 months of infection and often have high titres of neutralizing antibody in the cerebrospinal fluid (CSF) as well (Nathanson et al., 1979). Gudnad6ttir (1974) originally reported a single instance in which a virus isolate from an infected sheep could escape neutralization by the animal's own (autologous) serum. Narayan et al. (1977a, b, 1978) have conducted detailed studies on Hampshire and Border-Leicester sheep which confirmed and extended this observation. Antigenic variation is a potentially important observation because it could play a role in virus persistence and could provide a mechanism for the unpredictable appearance of focal CNS lesions. To determine the quantitative importance of antigenic drift, we have examined over 70 virus isolates obtained over 7 years from the blood (buffy coat), CSF or CNS of 20 Icelandic sheep infected with the 1514 strain of visna virus. Since Icelandic sheep develop neurological signs, which are rarely seen in most other breeds of sheep following visna infection, it was possible to /83/ $ SGM

2 1434 R. LUTLEY AND OTHERS relate antigenic variants to clinical disease. Our observations indicate that variant viruses occur at considerable frequency but do not appear essential to virus persistence or disease occurrence. METHODS Experimental animals and virus isolates. A group of 20 Icelandic sheep were inoculated intracerebrally with 106 TCDs0 of strain 1514 of visna virus, as described previously (P6tursson et al., 1976). Each sheep in this group was tested repeatedly for buffy coat virus in an explant-cocultivation system (P6tursson et al., 1976), and serial serum samples were also collected. These 20 sheep were studied for over 7 years, and animals developing severe illness were sacrificed and a diagnosis was made from clinical signs and a complete autopsy. At sacrifice, a standard set of CNS and other tissues was tested for virus by explantation and cocultivation (P6tursson et at., 1976). Table 1 summarizes the clinical course and virus isolations made from these animals. In addition, a group of five sheep were injected intracerebrally with 106 TCD~o of the 796 strain of visna virus, and 13 buffy coat isolates obtained from these sheep were included as a control on the typing procedure described below. Neutralization tests. Neutralization tests followed our published procedure (P6tursson et al., 1976). Each isolate was passed once in sheep choroid plexus (SCP) cultures, titrated and frozen at - 70 C. Tests were conducted by mixing an expected 10 ~ TCDso in 100 ~tl of diluent with 100 ~tl of serum dilution; mixtures were held at 4 C for 48 h and then inoculated into four SCP cultures (200 ~tl per culture) which were observed for 2 weeks. Simultaneous titration indicated that, in almost all tests, virus titres were between 1015 and TCDso. Replicate testing of selected sera or isolates indicated that typings were reproducible and serum titres repeatable within a twofold range. Typing with referenceant&era. All tests included two prototype viruses, strains 1514 and 796. Strain 1514 was the infecting virus and strain 796 was included because it is known to be serologically distinct. Although distinguish- Table 1. Clinical course, virus isolates, and antibody response in Icelandic intracerebrally with 106 TCDso of visna virus strain 1514 and followed for Neutralizing Virus isolates antibodyt Years to Diagnosis at, x ~ f. Sheep sacrifice sacrifice* Blood CSF CNS Serum CSF < Other < Visna Other Visna Visna (P):~ Other Visna ND ND "5 Visna 6 0 0(P) Visna 4 1 0(P) Other Other < Visna < Visna < Other 9 4 0(P) Other Totals sheep injected 7.5 years * Blank space indicates animal alive. Other diagnoses: 1519, traumatic tap; 1522, possible enterotoxaemia, 1525, paralysis of unknown cause; 1550, paratuberculosis; 1551, severe arthritis; 1556, traumatic tap; 1557, splenomegaly and debilitation. Blood isolates over 7-5 years; CSF isolates at 1 to 5, and 79 months. Neutralizing antibody titres to 1514 virus were measured and are shown as median of three to ten determinations at l to 4 years after infection. ~/P, Perfused. ND, Not determined.

3 Antigenic drift of visna virus 1435 Table 2. Typing of blood isolates from sheep infected with visna virus strains 1514 or 796." representative data using homotypic antisera Time of Typing antisera* In~cting isolate x, Antigenic virus Sheep (months) Divalent type Reference 128 < virus 1514 Reference < virus <4 > < <4 > < <4 <4 32 Neither 30 8 < < < <4 <4 32 Neither < < < <4 > < <4 <4 128 Neither <4 > < <4 > < < <4 > < <4 >250 > < * Typing antiserum: titre of typing antiserum against each virus isolate. able, the two strains were related, since 1514 was derived from 796 by multiple animal and SCP passages, as summarized by Narayan et al (1981). Using these two viruses, three typing sera were selected: serum 1514 (from a sheep infected with strain 1514) would neutralize 1514 but not 796; serum 796 (from a sheep infected with strain 796) would neutralize 796 but not 1514; divalent sera (from a sheep with a long-term infection with 1514 virus) would neutralize both viruses. An isolate was classified, according to its neutralization by the three reference sera, as 1514-like, 796-1ike, or like neither (Table 2). Tests with autologous sera. From the 20 sheep, a subgroup of seven animals was selected for autologous testing, with emphasis on animals yielding many isolates or animals developing clinical visna. From each such animal, serial sera, collected throughout a period of observation, were tested against the infecting strain 1514 and against a panel of autologous isolates from blood and CNS (where available). Neutralization tests followed the protocol described above. RESULTS A group of 20 Icelandic sheep were injected intracerebrally with 106 TCDso of visna virus, strain 1514, and followed for 7-5 years, with serial collection of blood and CSF for virus isolation and of serum for antibody determination. All sheep became infected as judged by virus isolation and serum antibody responses (Table 1). During the first 7 years of study, 149 blood isolates were made from 18 of the 20 animals (Table 1). From these 149 isolates, 59 were selected to represent 17 of 18 virus-positive sheep and the first 6 years of study; these 59 isolates were typed with reference antisera. In addition, blood

4 1436 R. LUTLEY AND OTHERS 151sl i I, 15191_.~ 1520~_..~ 1521~,.~) 1522~..~ 1523~.~,~.., ~N,~b,'N~l I d 1525 = [] [] 1557 t 0.5 [] [] r-i [33:3 I I I [] m (3(3 08) k~ R~ Time after infection (years) [] Fig. 1. Typing of 76 isolates from blood, CSF or CNS of 20 Icelandic sheep inoculated intracerebrally with 106 TCDs0 of visna virus strain 1514 and followed for 7 years. Typing was by neutralization with sera specific for 1514 and for 796 viruses. Squares indicate blood isolates, the circles CNS/CSF isolates; open symbols represent 1514-like serotype (64), the shaded symbols variant viruses (12) not neutralized by antiserum to 1514 virus. isolates from six sheep were tested with autologous sera, using one to seven isolates per animal (total of 24 isolates). As sheep developed severe illness, they were sacrificed and virus was isolated from CNS and other tissues. As summarized in Table 1, during the first 7-5 years of observation, 15 of 20 sheep were sacrificed, eight with clinical visna and seven with other diagnoses. CNS isolates were available from five animals with clinical visna and three with other diagnoses. A total of 17 CNS isolates were tested with reference antisera; 11 of these were also tested in the autologous system. Blood isolates Typing with reference antisera Table 2 shows representative results with prototype virus strains and with isolates from sheep infected with each of the prototype viruses. Several points are apparent. The reference typing sera clearly distinguished each prototype virus and did not cross-neutralize the heterotypic virus. Most isolates from 1514 virus-infected sheep were readily typed as 1514-like and isolates from 796 virus-infected sheep as 796-1ike. Finally, some isolates are classified as 'like neither' since they were not neutralized by either homotypic or heterotypic sera although they were neutralized by the divalent serum. Fig. 1 sets forth typing results on 20 sheep infected with strain 1514, for 59 blood isolates. About 15~ of isolates (9 of 59) are clearly different. All of the variant isolates exhibited a consistent pattern in that they were not neutralized by either homotypic serum but were neutralized by the divalent serum (see Table 2). It is apparent that the frequency of variants did not increase with time after infection. Also, variants are distributed widely among individual sheep, and when variants occurred they did not replace the parental virus. The one exception is sheep 1557, where the last three blood isolates were variants. CNS isolates Fig. 1 also presents data on i7 isolates from the CSF or CNS of 10 animals from the same

5 Antigenic drift of visna virus 1437 Table 3. Autologous neutralization tests on blood and CNS isolates from Icelandic sheep infected with visna virus strain 1514: sheep 1520, sacrificed at 2.5 years with clinical visna Isolate t" Source Years Blood CNS / Reference strain Sera, years after infection * > >500 >500 - > * Titre < 4. Table 4. Autologous neutralization tests on blood and CNS isolates from Icelandic sheep infected with visna virus strain 1514: sheep 1553, sacrificed at 7.2 years with clinical visna g Source Isolate Years Blood 0-3 CNS Reference strain at" Sera, years after infection * >250 > *, Titre <4. group of 20 infected sheep, including five animals with a clinical diagnosis of visna. Of these 17, three isolates were variants, and these came from sheep Of the total of 76 isolates typed by reference antisera, 12 (16~) were variants. However, antigenic drift (consistent isolation of variant viruses) was seen only for sheep Blood and CNS isolates Neutralization by autologous antisera To determine whether an infected sheep can neutralize the virus replicating in its own tissues, blood and/or CNS isolates were examined from seven sheep, of which five had clinical signs of visna. In each instance serial sera collected at intervals, between infection and death, were tested for neutralization titre against one to eight isolates from the same animal, and against 1514 and 796 viruses. Data for three representative sheep are shown in Tables, 3, 4 and 5. Tables 3 and 4 illustrate two animals (1520 and 1553) which failed to show antigenic variation. Serial sera neutralized all blood and CNS isolates just as well as the infecting strain Yet the potential of these sera to distinguish some variants is indicated by the failure of the serum from 1520 to neutralize reference strain 796. Table 5 shows that antigenic drift was clearly evident for sheep The firsf two blood isolates were neutralized but all of the subsequent six isolates from blood or CNS escaped neutralization. Autologous tests are summarized in Fig. 2 for seven animals and 35 isolates. Drift was seen only for sheep 1557, described above.

6 1438 R. LUTLEY AND OTHERS 1518 ~_].._] ' U ~ ) ' ' ' 1520[._1 d ,---~ e I [ [ I lss2l_l_j I I ] t I [--] ~ ~ Time after infection (years) k3 6 7 Fig. 2. Autologous neutralization tests on 35 isolates from blood, CSF or CNS of seven Icelandic sheep inoculated intracerebrally with 106 TCDso of visna virus strain 1514 and followed for 7 years. In autologous neutralization each isolate was tested against serial serum samples from the same sheep. Squares indicate blood isolates, the circles CSF/CNS isolates; open symbols represent isolates neutralized by autologous sera (29), the shaded symbols isolates not neutralized by autologous sera (6). Table 5. Autologous neutralization tests on blood and CNS isolates from Icelandic sheep infected with visna virus strain 1514: sheep 1557, sacrificed at 6"6 years with clinical visna Isolate Sera, years after infection g "~ "3 Source Years l Blood 0.5 * > >32 > CNS Reference strain * Titre <4. DISCUSSION The use of sera readily capable of distinguishing the two antigenic variants (1514 and 796) provides evidence of the specificity of the typing system. The system is further authenticated by the observation that most sheep yielded isolates consistent with the infecting serotype. The fact that all variant viruses consistently escaped neutralization by both homotypic reference sera but were neutralized by the divalent serum (obtained from a long-term infected sheep) suggests that the divalent serum recognized additional antigenic determinant(s) which are shared by prototype and variant viruses. The methods used in this study are relatively insensitive, since the homotypic typing sera are polyclonal and may fail to identify minimal antigenic variants. The use of monoclonal antibodies might reveal a higher frequency of variants, since monoclones can distinguish single amino acid changes in neutralizing glycoproteins (Laver et al., 1979a, b). Because the 1514 strain was not plaque-purified, it could well have included a minority population of antigenic variants. Furthermore, studies of influenza and rabies viruses with monoclonal antibodies (Gerhard et al., 1980; Wiktor & Koprowski, 1980) show that even cloned viruses always contain a minority population (of the order of 10 -s to 10-6) of each specific antigenic variant. The data of Clements et al. (1980) and of Scott et al. (1979) indicate that antigenic variants of visna virus differ from their respective parents by one or more point

7 Antigenic drift of visna virus 1439 mutations. Thus, it is likely that variants are constantly being generated and are present in all stocks of visna virus. These considerations suggest that long-term animal infection provides a system for the selection of variant viruses rather than for their production. The finding that 16~ of isolates register as variants using a relatively insensitive typing system indicates an increase in their frequency during infection, since the parent 1514 stock contained much less than 1 ~ of variants as evidenced by the consistent ability of the typing sera to neutralize at least 1000 TCDs0 of 1514 virus. Two possible roles have been suggested (Narayan et al., 1977a, b, 1978) for visna virus variants in pathogenesis: as a mechanism for virus persistence, and as an explanation of lesion initiation. The present study, has, for the first time, provided data on the frequency of antigenic variants isolated over many years from one group of animals. Although the number of variants is substantial, there is no evidence that they replace the infecting serotype with the passage of time. Rather, parental and variant viruses appear to co-exist, suggesting that antigenic drift is not essential for virus persistence. Tests of 35 isolates with autologous sera confirmed this view. Since these 35 isolates were derived from cells constantly immersed in antibody-bearing fluids, this should have provided an optimal milieu for the selection of non-neutralizable variant viruses. Our findings resemble the observations of Narayan et al. (1978) who infected Hampshire and Border-Leicester sheep and found that, in two of seven animals studied, a number of buffy coat isolates were capable of totally escaping neutralization by autologous serum obtained at the time of, and for some interval after, virus isolation. It is clear that Icelandic sheep are more permissive for visna virus (P6tursson et al., 1976; P~lsson et al., 1977; Georgsson et al., 1978) than the sheep used by Narayan et al. (1974, 1977b). Infected Icelandic sheep may develop a broad serum-neutralizing response more rapidly (Henle & Lief, 1963) thereby limiting the replication of variant viruses (Narayan et al., 1981). The onset and course of clinical disease is notoriously variable and unpredictable in visna. This is confirmed in the present study in which 20 sheep have been followed for over 7 years with eight animals sacrificed with clinical disease at 3, 22, 30, 67, 78, 78, 86 and 90 months, respectively. Furthermore, our recent pathological observations (Georgsson et al., 1982) clearly indicate that individual sheep may simultaneously bear recent and old demyelinating lesions in various stages of evolution. Narayan et al. (1977a, 1978) and Clements et al. (1980) have suggested that this irregular course of CNS disease might be explained by the appearance in the CNS at unpredictable intervals of antigenic variants capable of temporarily escaping neutralization. We have now examined, by homotyping, 17 CNS and CSF isolates and, with autologous antiserum, 11 CNS isolates. The frequency of variant viruses (three of 17 isolates, or 18 ~) is similar to that seen with blood isolates (15~o). Although a majority of CNS isolates were made from sheep with clinical visna, all the variant isolates came from an animal without clinical visna. From these observations, we are forced to conclude that, although antigenic variants are preferentially selected in the CNS as in the blood, they do not appear to play an essential role in the evolution of CNS lesions. Similar findings and interpretations are being concurrently reported by Thormar et al. (1983). The expert technical assistance of Kolbrun Kistinsdottir, Noelle Naudot, and Sigurbjorg Thorsteinsdottir is gratefully acknowledged. This work was supported in part by USPHS grant NS REFERENCES CLEMENTS, J. E., PEDERSON, F. S., NARAYAN, O. & HASELTINE, W. S. (1980). Genomic changes associated with antigenic variation of visna virus during persistent infection. Proceedings of the National Academy of Sciences, U.S.A. 77, GEORGSSON, G., PI~TURSSON, G., PALSSON, P. A., MILLER, A. & NATHANSON, N. (1978). Experimental visna in fetal Icelandic sheep. Journal of Comparative Pathology 88, GEORGSSON, G., MARTIN, J. R., KLEIN, J., P,~LSSON, P. A., NATHANSON, N. & PI~TURSSCgN, G. (1982). Primary demyelination in visna. An ultrastructural study of Icelandic sheep with clinical signs following experimental infection. Acta neuropathologica 57,

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