VIROLOGY
64, 415-420 (1975)
Antigenic
Relationships
Between
Strains
of Tobacco
Mosaic
Virus
M. H. V. VAN REGENMORTEL Department
of Microbiology, Accepted
University
of Cape Town, South Africa
October 29, 1974
The serological relationships between six strains of tobacco mosaic virus were studied with antisera from 40 rabbits bled at regular intervals during at least 8 mo. The extent of cross reactivity between strains was expressed by a serological differentiation index (SDI) equal to the difference between homologous and heterologous titers denoted as Neg Log,. The extent of cross reactivity between two strains was determined in reciprocal serological tests, using antisera against each of the two strains. Average SD1 values calculated from a large number of bleedings taken from different animals agreed closely with the corresponding SD1 values calculated from reciprocal serological tests. The intraclass correlation coefficient between two sets of SD1 values obtained in reciprocal serological tests was r’ = 0.95. A correlation coefficient of r = 0.75 was calculated between the serological relatedness expressed as SD1 values and the extent of sequence homology in the coat protein of different strains. INTRODUCTION
Serological cross reactions have been extensively used to establish the degree of similarity between viruses. However, when attempts are made to quantify with some precision the extent of cross reactivity between two antigens, considerable difficulties arise. Rabbits are most often used for this type of study and in the absence of inbred strains it is common to observe very large differences in the individual responses of different animals. The route and number of injections used for immunization as well as the time chosen to bleed the animal also greatly influence the apparent cross reactivity revealed by different antisera (Allen, 1968; Koenig and Bercks, 1968; Kassanis and Phillips, 1970). In a previous study of the serological relationship between tobacco mosaic virus (TMV) and cucumber virus 4 (van Regenmortel and von Wechmar, 1970) different antisera obtained from 18 rabbits showed such large differences in apparent cross reactivity that it seemed arbitrary to decide whether the two viruses were closely or distantly related. It must be stressed that 415 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
this conclusion was based on data obtained at the height of the immune response, i.e., at a point in time when the maximum homologous titer had been reached. Since cross reactivities may differ greatly at different stages of the immunization process, an attempt has been made in the present work to express the degree of serological relationship between different antigens in terms of the average cross reactivity observed in different antisera collected over an extended period of immunization. The degree of serological relatedness between six serotypes of tobacco mosaic virus has been examined in the present study. The amino acid sequence of the coat protein of four of the serotypes is known (common TMV; Y-TAMV; G-TAMV, HR; Hennig and Wittmann, 1972) as well as the amino acid composition of the other two serotypes (CV4; van Regenmortel, 1967; CGMMV-C: Kurachi et al., 1972; Nozu et al., 1971). It was felt that the use of well-characterized virus strains would help to clarify the controversy regarding the validity of comparative serological studies, since a correlation between serological and biochemical data could also be attempted
416
M. H. V. VAN REGENMORTEL
in this case. It has been argued previously (van Regenmortel, 1966; 1967; van Regenmortel and von Wechmar, 1970) that the variability of serological data precludes the use of serology for defining degrees of relatedness between virus strains. On the other hand, Koenig and Bercks (1968) and Koenig and Givord (1974) have shown that in spite of the variability of heterologous titers, it is possible by using a large number of bleedings from different animals, to obtain an approximate estimate of the extent of cross reactivity between different viruses. The present work confirms that meaningful serological comparisons between virus strains are possible provided a sufficient number of bleedings from different animals are examined. A preliminary report of the results has been published (van Regenmortel and von Wechmar, 1974). MATERIALS
AND
METHODS
The TMV strains used in previous studies (van Regenmortel, 1967) were used again in this work. They are = TMV, common strain; Y-TAMV, yellow tomato atypical mosaic strain; G-TAMV, green tomato atypical mosaic strain; CV4, cucumber virus 4; HR, Holmes’ ribgrass strain. Strain Y-TAMV is serologically indistinguishable from strain Dahlemense while G-TAMV is serologically identical with strain U2 (see Hennig and Wittmann, 1972; van Regenmortel, 1967). An isolate of cucumber green mottle mosaic virus (CGMMV-C) obtained from Dr. T. Inouye was also included in the study. A partial amino acid sequence of this strain has been published (Kurachi et al., 1972). All the strains were purified by precipitation with polyethylene glycol as described previously. (von Wechmar and van Regenmortel, 1970). A total of 40 rabbits were injected; 10 with CV4,8 with TMV, 6 with G-TAMV, 6 with Y-TAMV, 5 with CGMMV-C, and 5 with HR. All the animals received three intramuscular injections of 5 mg of virus in Freund’s incomplete adjuvant, the last two injections being given 14 and 25 wk after
the first one. The development of homologous and heterologous titers was determined by analyzing antisera collected from the rabbits at various intervals over a period of at least 8 mo. Precipitin tests were performed in tubes kept in a water bath at 40” for 3 hr; serial 2-fold dilutions of antiserum were mixed with 0.01 mg/ml antigen. Final readings of the precipitates were taken after the tubes had been left undisturbed at room temperature for 18 hr. The extent of cross-reactivity between two serotypes was expressed by a serological differentiation index (SDI) (van Regenmortel and von Wechmar, 1970) defined as the number of 2-fold dilution steps separating homologous and heterologous titers. SD1 values are equal to the difference in these titers expressed as Neg Log,. RESULTS
The development of homologous and heterologous titers over a period of 8 mo in two rabbits injected with strains Y-TAMV and G-TAMV, respectively, is illustrated in Figs. 1 and 2. SD1 values for these two antisera are shown in Table I. It can be seen that with the exception of the first 3 wk, SD1 values remain fairly constant throughout the entire immunization period. Lower SD1 values at the early stages of immunization were a fairly regular feature of the antisera studied in the present investigation. Such changes in SD1 were especially marked in the case of more distantly related strains, e.g., Y-TAMV and CV4 (Table I). To obtain average SD1 values that are more representative of the situation existing during long periods of immunization, the titers obtained during the first 3 wk of immunization were not taken into account. In a number of instances, late in the immunization process heterologous titers decreased more rapidly than homologous ones (see Table I, antiserum G-TAMV). However, this was not a regular feature, and was not characteristic for any particular antiserum or antigen. Representative SD1 values describing the extent of cross reactivity between two serotypes were obtained by averaging the val-
ANTIGENIC
0
2
RELATIONSHIPS
417
OF TMV STRAINS
4 MONTHS
6
6
FIG. 1. Development of homologous and heterologous titers of a Y-TAMV antiserum. 0-O Y-TAMV; Cl---U TMV; O---O G-TAMV; A-A CV4. The animal received three intramuscular injections at times shown by the arrows.
MONTHS
FIG. 2. Development of homologous and heterologous titers of a G-TAMV antiserum. O-O G-TAMV; O----O Y-TAMV; Cl-0 TMV; A-A CV4. The animal received three intramuscular injections at times shown by the arrows.
ues from at least five animals that had been bled 8-12 times over a period of at least 8 mo. The results of all the cross-reactions obtained with 14 pairs of serotypes are summarized in Table 2. The extent of cross reactivity between two strains was determined in reciprocal serological tests, using antisera against each of the two strains. The most important finding that came to light from these results was the fact that average reciprocal SD1 values showed remarkable agreement (Table 3). No significant difference between reciprocal SD1 values was calculated in a x2 test at the 0.5% level. The intraclass correlation coefficient between the two sets of reciprocal SD1
values was r’ = 0.95. It was, therefore, possible to use average SD1 values from reciprocal tests as an index of the serological cross reactivity between two serotypes. When the degree of serological relatedness between some of the serotypes is compared to the extent of sequence homology in their coat proteins (Table 3), it appears that there is some correlation between the two (r = 0.75). By means of a t test, this correlation was found to be significant at a level P = 0.1. This is surprising in view of the fact that only a limited number of amino acid residues of the coat protein are contributing to the antigenic determinants of the virus (Sengbusch, 1965, van Regenmortel, 1967).
418
M. H. V. VAN REGENMORTEL TABLE
I
CROSS REACTIVITIES BETWEEN SERO??IPESOF TOBACCO MOSAIC VIRUS Antiserum
Anti Y-TAMV
Anti G-TAMV
Number of weeks after first injection
Reciprocal of homologous precipitin titer
2 3 4 9 12 14 16 18 26 28 31 32
128 256 512 512 256 512 256 128 2048 1024 512 512
7 8 9 9 8 9 8 7 11 10 9 9
2 0 0 1 1 1 1 1 2 2 1 1
2 3 4 8 9 12 14 16 18 26 28 33
128 256 512 256 256 128 2048 2048 4096 4096 4096 2048
7 8 9 8 8 7 11 11 12 12 12 11
1 2 4 3 3 2 3 2 3 4 4 3
o SD1 values represent the difference
Homologous titer in Neg Log,
SDI” with TMV
between homologous TABLE
1 1 1 2 3 2 2 2 3 3 3 3
SD1 with Y-TAMV
2 3 4 6 7 6 5 4 8 7
-
7 1 2 3 4 4 3 4 5 7 8 8
1 2 2 2 2 1 2 1 2 2 3 2
titers expressed as Neg Log,.
SEROTYPES,OBTAINED IN CROSS-REACTIONTEST@ Antiserum
Anti-GTAMV G-TAMV Y-TAMV TMV HR cv4 CGMMV-C
and heterologous
SD1 with cv4
2
SEROLOGICALDIFFERENTIATION INDICES (SDI)” BETWEEN TMV Antigen
SD1 with G-TAMV
1.8 2.8 5.1 5.8
0 zt 1.0 zt 0.8 * 1.4 * 1.1
Anti-YTAMV
Anti-TMV
1.9 i 0.8 0 1.1 * 0.7 3.7 * 0.8 6.1 +z 1.4
2.5 h 0.8 1.2 + 0.8 0 2.1 * 1.0 3.8 * 1.0
Anti-HR
AntiCV4
3.8 & 1.2 4.3 * 0.7 2.1 * 0.8 0 4.6 + 1.1
6.4 5.7 4.5 4.9
zt 0.9 * 1.0 * 1.2 i 1.3 0 5.9 * 1.2
AntiCGMMV-C 6.7 * 0.9 7.0 * 1.1 7.5 * 1.4 6.5 + 1.1 0
0 SD1 values represent the difference between homologous and heterologous titers expressed as Neg Log, + standard deviation. b Each SD1 value represents an average calculated from a minimum of 40 bleedings obtained from at least five rabbits. DISCUSSION
The main purpose of the present work was to ascertain whether the well-documented variability of serological data pre-
eludes the use of serology for defining degrees of relationship between viruses. The results show that meaningful serological comparisons between TMV serotypes are possible, provided a sufficient number
ANTIGENIC
RELATIONSHIPS
419
OF TMV STRAINS
TABLE 3 SEROLOGICALRELATIONSHIPSAND SEQUENCEHOMOLOGY BETWEEN TMV SEROT~PES
Antiserum
Test antigen
SDI”
Anti-TMV Anti-Y-TAMV Anti-TMV Anti-TMV Anti-Y-TAMV Anti-TMV Anti-G-TAMV Anti-HR Anti-Y-TAMV Anti-G-TAMV Anti-CV4 Anti-CGMMV-C Anti-CGMMV-C Anti-CGMMV-C
Y-TAMV G-TAMV HR G-TAMV HR cv4 HR cv4 cv4 cv4 CGMMV-C G-TAMV Y-TAMV TMV
1.2 1.9 2.1 2.5 3.7 3.8 5.1 4.6 6.1 5.8 5.9 6.7 7.0 7.5
SD1 in reciprocal testa. b 1.1 1.8 2.1 2.8 4.3 4.5 3.8 4.9 5.7 6.4 6.5
AEige
1.2 1.9 2.1 2.7 4.0 4.2 4.5 4.8 5.9 6.1 6.2
Percentage difference in sequence’, d 18 30 56 26 53 54
“Data from Table 2. SD1 values represent the difference between homologous and heterologous titers expressed as Neg Log,. bNo significant difference at the 0.5% level between the two reciprocal sets of data was observed on the basis of a x1 test. The intraclass correlation coefficient between the two sets of reciprocal SD1 values was r’ = 0.95. ‘The correlation coefficient between these data and the average SD1 values was r = 0.75. dData from Hennig and Wittmann (1972).
of bleedings from different animals are examined. Variations in the degree of apparent cross reactivity exhibited by different antisera may arise from differences between individual animals (Bercks, 1963; van Regenmortel and von Wechmar, 1970), from the route and number of injections used for immunization (Hollings and Stone, 1965) or from the time of bleeding (Allen, 1968; Koenig and Bercks, 1968). To minimize these sources of variation in the present work, all animals received the same dose of immunogen and number of intramuscular injections in adjuvant and they were bled at regular intervals over a minimum period of 8 mo. Some workers have reported marked increases in cross reactivity between plant viruses in successive bleedings from the same animal (Tremaine and Wright, 1967; Kassanis and Phillips, 1970) while others have reported decreases or increases with time, depending on the particular antigen or animal used (Koenig and Bercks, 1968; Koenig and Givord, 1974). One valid generalization which seems to apply is that
differences between animals and successive bleedings are minimized by averaging cross reactivities observed over a fairly long period of time. Since in the present work large individual differences in SD1 values were found mainly in the early stages of immunization, cross-reaction data pertaining to the first 3 wk of immunization were not included in the calculation of averaged SD1 values (Table 2). The results summarized in Table 3 show that when the data from a sufficient number of bleedings collected from at least five rabbits are averaged, the degrees of relatedness between serotypes calculated from reciprocal serological tests agree very closely (r' = 0.95). This excellent agreement between reciprocal tests is in sharp contrast with the previously reported lack of reciprocity obtained in individual cases (van Regenmortel, 1967; van Regenmortel and von Wechmar, 1970) and it emphasizes again the danger of basing conclusions on too few bleedings from too few animals. The results in Tables 2 and 3 show that a continuous range of increasingly distantly related serotypes of TMV exist and that it
420
M. H. V. VAN REGENMORTEL
would be arbitrary to distinguish between close and distant serological relationships in this case. A distinction between strains and serotypes based on the extent of crossreactivity (Babos and Kassanis, 1963; Kassanis and Phillips, 1970) seems impractical and the present study adds further weight to the suggestion of van Regenmortel and von Wechmar (1970) that the term “serotype” be used to denote any virus strain that is serologically distinguishable from other strains, regardless of whether the serological differences are large or small. The degree of serological relationship between viruses measures indirectly the extent of homology in the amino acid sequence of the corresponding coat proteins. In the case of TMV it is well-known that only a limited number of amino acid residues of the protein contribute to the antigenic determinants of the virus (see van Regenmortel, 1966; 1967). The results in Table 3 show that there is, nevertheless, some correlation between sequence homology and the extent of serological relationship. Furthermore, strain CGMMV-C which has the greatest number of net amino acid exchanges compared to common TMV (30 exchanges) and which appears to have the most divergent sequence (Kurachi et al., 1972) also shows the lowest degree of serological cross-reactivity with TMV. ACKNOWLEDGMENTS The author gratefully acknowledges the skillful assistance of Barbara von Wechmar and Nelly Lelarge. REFERENCES ALLEN, W. R. (1968). Tomato bushy stunt virus from Prunes auium II. Serological typing and the characterization of antibody types and activities. Can.J. Bot. 46, 229-233. BABOS, P., and KASSANIS, B. (1963). Serological relationships and some properties of tobacco necrosis virus strains. J. Gen. Microbial. 32, 135-144. BERCKS, R. (1963). Untersuchungen tiber individuelle Unterschiede von Antiseren gegen Kartoffel X-virus
bei Reaktionen mit verwandten Viren. Phytopathol. z. 47, 301-313. HENNIC B., and W~T~MANN, H. G. (1972). Tobacco mosaic virus = mutants and strains. In “Principles and Techniques in Plant Virology” (C. I. Kado and H. 0. Agrawal, eds.), pp. 546-594. Van Nostrand Reinhold, New York. HOLLINGS, M., and STONE, 0. M. (1965). Studies of pelargonium leaf curl virus II. Relationships to tomato bushy stunt and other viruses. Ann. Appl. Biol. 56, 87-98. KASSANIS, B., and PHILLIPS, M. P. (1970). Serological relationships of strains of tobacco necrosis virus and their ability to activate strains of satellite virus. J. Gen. Viral. 9, 119-126. KOENIG, R., and BERCKS, R. (1968). .&nderungen im heterologen Reaktionsvermogen von Antiseren gegen Vertreter der potato virus X-gruppe im Laufe des Immunisierungsprozesses. Phytopathol. Z. 61, 382-398. KOENIG, R., and GIVORD, L. (1974). Serological interrelationships in the turnip yellow mosaic virus group. virology 58, 119-125. KURACHI, K., FUNATSU, G., FUNATSU, M., and HIDAKA, S. (1972). Partial amino acid sequence of cucumber green mottle mosaic virus coat protein. Agr. Biol. Chem. 36, 1109-1116. Nozu, Y., TOCHIHARA, H., KOMURO, Y., and OKADA, Y. (1971). Chemical and immunological characterization of cucumber green mottle mosaic virus (watermelon strain) protein. Virology 45, 577-585. SENGBUSCH, P. von (1965). Aminoslureaustausche und Tertilrstruktur eines Protein. Vergleich von Mutanten des Tabakmosaikvirus mit serologischen und physikochemischen Methoden. Z. Vererbungslehre 96,364-386. TREMAINE, J. H., and WRIGHT, N. S. (1967). Crossreactive antibodies in antisera to two strains of southern bean mosaic virus. Virology 31, 481-488. VAN R~GENMORTEL, M. H. V. (1966). Plant virus serology. Advan. Virus Res. 12, 207-271. VAN REGENMORTEL,M. H. V. (1967). Serological studies on naturally occurring strains and chemically induced mutants of tobacco mosaic virus. Virology 31, 467-480. VAN REGENMORTEL, M. H. V., and VON WECHMAR, M. B. (1970). A reexamination of the serological relationship between tobacco mosaic virus and cucumber virus 4. Virology 41, 330-338. VAN REGENMORTEL, M. H. V., and VON WECHMAR, M. V. (1974). Serological relationships of strains of tobacco mosaic virus. S. Ajr. J. Sci. 70, 59-60. VON WECHMAR, M. B., and VAN REGENMORTEL, M. H. V. (1970). A simple procedure for purifying tobacco mosaic virus strains. S. Afr. Med. J. 44, 151.
64, 415-420 (1975)
Antigenic
Relationships
Between
Strains
of Tobacco
Mosaic
Virus
M. H. V. VAN REGENMORTEL Department
of Microbiology, Accepted
University
of Cape Town, South Africa
October 29, 1974
The serological relationships between six strains of tobacco mosaic virus were studied with antisera from 40 rabbits bled at regular intervals during at least 8 mo. The extent of cross reactivity between strains was expressed by a serological differentiation index (SDI) equal to the difference between homologous and heterologous titers denoted as Neg Log,. The extent of cross reactivity between two strains was determined in reciprocal serological tests, using antisera against each of the two strains. Average SD1 values calculated from a large number of bleedings taken from different animals agreed closely with the corresponding SD1 values calculated from reciprocal serological tests. The intraclass correlation coefficient between two sets of SD1 values obtained in reciprocal serological tests was r’ = 0.95. A correlation coefficient of r = 0.75 was calculated between the serological relatedness expressed as SD1 values and the extent of sequence homology in the coat protein of different strains. INTRODUCTION
Serological cross reactions have been extensively used to establish the degree of similarity between viruses. However, when attempts are made to quantify with some precision the extent of cross reactivity between two antigens, considerable difficulties arise. Rabbits are most often used for this type of study and in the absence of inbred strains it is common to observe very large differences in the individual responses of different animals. The route and number of injections used for immunization as well as the time chosen to bleed the animal also greatly influence the apparent cross reactivity revealed by different antisera (Allen, 1968; Koenig and Bercks, 1968; Kassanis and Phillips, 1970). In a previous study of the serological relationship between tobacco mosaic virus (TMV) and cucumber virus 4 (van Regenmortel and von Wechmar, 1970) different antisera obtained from 18 rabbits showed such large differences in apparent cross reactivity that it seemed arbitrary to decide whether the two viruses were closely or distantly related. It must be stressed that 415 Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
this conclusion was based on data obtained at the height of the immune response, i.e., at a point in time when the maximum homologous titer had been reached. Since cross reactivities may differ greatly at different stages of the immunization process, an attempt has been made in the present work to express the degree of serological relationship between different antigens in terms of the average cross reactivity observed in different antisera collected over an extended period of immunization. The degree of serological relatedness between six serotypes of tobacco mosaic virus has been examined in the present study. The amino acid sequence of the coat protein of four of the serotypes is known (common TMV; Y-TAMV; G-TAMV, HR; Hennig and Wittmann, 1972) as well as the amino acid composition of the other two serotypes (CV4; van Regenmortel, 1967; CGMMV-C: Kurachi et al., 1972; Nozu et al., 1971). It was felt that the use of well-characterized virus strains would help to clarify the controversy regarding the validity of comparative serological studies, since a correlation between serological and biochemical data could also be attempted
416
M. H. V. VAN REGENMORTEL
in this case. It has been argued previously (van Regenmortel, 1966; 1967; van Regenmortel and von Wechmar, 1970) that the variability of serological data precludes the use of serology for defining degrees of relatedness between virus strains. On the other hand, Koenig and Bercks (1968) and Koenig and Givord (1974) have shown that in spite of the variability of heterologous titers, it is possible by using a large number of bleedings from different animals, to obtain an approximate estimate of the extent of cross reactivity between different viruses. The present work confirms that meaningful serological comparisons between virus strains are possible provided a sufficient number of bleedings from different animals are examined. A preliminary report of the results has been published (van Regenmortel and von Wechmar, 1974). MATERIALS
AND
METHODS
The TMV strains used in previous studies (van Regenmortel, 1967) were used again in this work. They are = TMV, common strain; Y-TAMV, yellow tomato atypical mosaic strain; G-TAMV, green tomato atypical mosaic strain; CV4, cucumber virus 4; HR, Holmes’ ribgrass strain. Strain Y-TAMV is serologically indistinguishable from strain Dahlemense while G-TAMV is serologically identical with strain U2 (see Hennig and Wittmann, 1972; van Regenmortel, 1967). An isolate of cucumber green mottle mosaic virus (CGMMV-C) obtained from Dr. T. Inouye was also included in the study. A partial amino acid sequence of this strain has been published (Kurachi et al., 1972). All the strains were purified by precipitation with polyethylene glycol as described previously. (von Wechmar and van Regenmortel, 1970). A total of 40 rabbits were injected; 10 with CV4,8 with TMV, 6 with G-TAMV, 6 with Y-TAMV, 5 with CGMMV-C, and 5 with HR. All the animals received three intramuscular injections of 5 mg of virus in Freund’s incomplete adjuvant, the last two injections being given 14 and 25 wk after
the first one. The development of homologous and heterologous titers was determined by analyzing antisera collected from the rabbits at various intervals over a period of at least 8 mo. Precipitin tests were performed in tubes kept in a water bath at 40” for 3 hr; serial 2-fold dilutions of antiserum were mixed with 0.01 mg/ml antigen. Final readings of the precipitates were taken after the tubes had been left undisturbed at room temperature for 18 hr. The extent of cross-reactivity between two serotypes was expressed by a serological differentiation index (SDI) (van Regenmortel and von Wechmar, 1970) defined as the number of 2-fold dilution steps separating homologous and heterologous titers. SD1 values are equal to the difference in these titers expressed as Neg Log,. RESULTS
The development of homologous and heterologous titers over a period of 8 mo in two rabbits injected with strains Y-TAMV and G-TAMV, respectively, is illustrated in Figs. 1 and 2. SD1 values for these two antisera are shown in Table I. It can be seen that with the exception of the first 3 wk, SD1 values remain fairly constant throughout the entire immunization period. Lower SD1 values at the early stages of immunization were a fairly regular feature of the antisera studied in the present investigation. Such changes in SD1 were especially marked in the case of more distantly related strains, e.g., Y-TAMV and CV4 (Table I). To obtain average SD1 values that are more representative of the situation existing during long periods of immunization, the titers obtained during the first 3 wk of immunization were not taken into account. In a number of instances, late in the immunization process heterologous titers decreased more rapidly than homologous ones (see Table I, antiserum G-TAMV). However, this was not a regular feature, and was not characteristic for any particular antiserum or antigen. Representative SD1 values describing the extent of cross reactivity between two serotypes were obtained by averaging the val-
ANTIGENIC
0
2
RELATIONSHIPS
417
OF TMV STRAINS
4 MONTHS
6
6
FIG. 1. Development of homologous and heterologous titers of a Y-TAMV antiserum. 0-O Y-TAMV; Cl---U TMV; O---O G-TAMV; A-A CV4. The animal received three intramuscular injections at times shown by the arrows.
MONTHS
FIG. 2. Development of homologous and heterologous titers of a G-TAMV antiserum. O-O G-TAMV; O----O Y-TAMV; Cl-0 TMV; A-A CV4. The animal received three intramuscular injections at times shown by the arrows.
ues from at least five animals that had been bled 8-12 times over a period of at least 8 mo. The results of all the cross-reactions obtained with 14 pairs of serotypes are summarized in Table 2. The extent of cross reactivity between two strains was determined in reciprocal serological tests, using antisera against each of the two strains. The most important finding that came to light from these results was the fact that average reciprocal SD1 values showed remarkable agreement (Table 3). No significant difference between reciprocal SD1 values was calculated in a x2 test at the 0.5% level. The intraclass correlation coefficient between the two sets of reciprocal SD1
values was r’ = 0.95. It was, therefore, possible to use average SD1 values from reciprocal tests as an index of the serological cross reactivity between two serotypes. When the degree of serological relatedness between some of the serotypes is compared to the extent of sequence homology in their coat proteins (Table 3), it appears that there is some correlation between the two (r = 0.75). By means of a t test, this correlation was found to be significant at a level P = 0.1. This is surprising in view of the fact that only a limited number of amino acid residues of the coat protein are contributing to the antigenic determinants of the virus (Sengbusch, 1965, van Regenmortel, 1967).
418
M. H. V. VAN REGENMORTEL TABLE
I
CROSS REACTIVITIES BETWEEN SERO??IPESOF TOBACCO MOSAIC VIRUS Antiserum
Anti Y-TAMV
Anti G-TAMV
Number of weeks after first injection
Reciprocal of homologous precipitin titer
2 3 4 9 12 14 16 18 26 28 31 32
128 256 512 512 256 512 256 128 2048 1024 512 512
7 8 9 9 8 9 8 7 11 10 9 9
2 0 0 1 1 1 1 1 2 2 1 1
2 3 4 8 9 12 14 16 18 26 28 33
128 256 512 256 256 128 2048 2048 4096 4096 4096 2048
7 8 9 8 8 7 11 11 12 12 12 11
1 2 4 3 3 2 3 2 3 4 4 3
o SD1 values represent the difference
Homologous titer in Neg Log,
SDI” with TMV
between homologous TABLE
1 1 1 2 3 2 2 2 3 3 3 3
SD1 with Y-TAMV
2 3 4 6 7 6 5 4 8 7
-
7 1 2 3 4 4 3 4 5 7 8 8
1 2 2 2 2 1 2 1 2 2 3 2
titers expressed as Neg Log,.
SEROTYPES,OBTAINED IN CROSS-REACTIONTEST@ Antiserum
Anti-GTAMV G-TAMV Y-TAMV TMV HR cv4 CGMMV-C
and heterologous
SD1 with cv4
2
SEROLOGICALDIFFERENTIATION INDICES (SDI)” BETWEEN TMV Antigen
SD1 with G-TAMV
1.8 2.8 5.1 5.8
0 zt 1.0 zt 0.8 * 1.4 * 1.1
Anti-YTAMV
Anti-TMV
1.9 i 0.8 0 1.1 * 0.7 3.7 * 0.8 6.1 +z 1.4
2.5 h 0.8 1.2 + 0.8 0 2.1 * 1.0 3.8 * 1.0
Anti-HR
AntiCV4
3.8 & 1.2 4.3 * 0.7 2.1 * 0.8 0 4.6 + 1.1
6.4 5.7 4.5 4.9
zt 0.9 * 1.0 * 1.2 i 1.3 0 5.9 * 1.2
AntiCGMMV-C 6.7 * 0.9 7.0 * 1.1 7.5 * 1.4 6.5 + 1.1 0
0 SD1 values represent the difference between homologous and heterologous titers expressed as Neg Log, + standard deviation. b Each SD1 value represents an average calculated from a minimum of 40 bleedings obtained from at least five rabbits. DISCUSSION
The main purpose of the present work was to ascertain whether the well-documented variability of serological data pre-
eludes the use of serology for defining degrees of relationship between viruses. The results show that meaningful serological comparisons between TMV serotypes are possible, provided a sufficient number
ANTIGENIC
RELATIONSHIPS
419
OF TMV STRAINS
TABLE 3 SEROLOGICALRELATIONSHIPSAND SEQUENCEHOMOLOGY BETWEEN TMV SEROT~PES
Antiserum
Test antigen
SDI”
Anti-TMV Anti-Y-TAMV Anti-TMV Anti-TMV Anti-Y-TAMV Anti-TMV Anti-G-TAMV Anti-HR Anti-Y-TAMV Anti-G-TAMV Anti-CV4 Anti-CGMMV-C Anti-CGMMV-C Anti-CGMMV-C
Y-TAMV G-TAMV HR G-TAMV HR cv4 HR cv4 cv4 cv4 CGMMV-C G-TAMV Y-TAMV TMV
1.2 1.9 2.1 2.5 3.7 3.8 5.1 4.6 6.1 5.8 5.9 6.7 7.0 7.5
SD1 in reciprocal testa. b 1.1 1.8 2.1 2.8 4.3 4.5 3.8 4.9 5.7 6.4 6.5
AEige
1.2 1.9 2.1 2.7 4.0 4.2 4.5 4.8 5.9 6.1 6.2
Percentage difference in sequence’, d 18 30 56 26 53 54
“Data from Table 2. SD1 values represent the difference between homologous and heterologous titers expressed as Neg Log,. bNo significant difference at the 0.5% level between the two reciprocal sets of data was observed on the basis of a x1 test. The intraclass correlation coefficient between the two sets of reciprocal SD1 values was r’ = 0.95. ‘The correlation coefficient between these data and the average SD1 values was r = 0.75. dData from Hennig and Wittmann (1972).
of bleedings from different animals are examined. Variations in the degree of apparent cross reactivity exhibited by different antisera may arise from differences between individual animals (Bercks, 1963; van Regenmortel and von Wechmar, 1970), from the route and number of injections used for immunization (Hollings and Stone, 1965) or from the time of bleeding (Allen, 1968; Koenig and Bercks, 1968). To minimize these sources of variation in the present work, all animals received the same dose of immunogen and number of intramuscular injections in adjuvant and they were bled at regular intervals over a minimum period of 8 mo. Some workers have reported marked increases in cross reactivity between plant viruses in successive bleedings from the same animal (Tremaine and Wright, 1967; Kassanis and Phillips, 1970) while others have reported decreases or increases with time, depending on the particular antigen or animal used (Koenig and Bercks, 1968; Koenig and Givord, 1974). One valid generalization which seems to apply is that
differences between animals and successive bleedings are minimized by averaging cross reactivities observed over a fairly long period of time. Since in the present work large individual differences in SD1 values were found mainly in the early stages of immunization, cross-reaction data pertaining to the first 3 wk of immunization were not included in the calculation of averaged SD1 values (Table 2). The results summarized in Table 3 show that when the data from a sufficient number of bleedings collected from at least five rabbits are averaged, the degrees of relatedness between serotypes calculated from reciprocal serological tests agree very closely (r' = 0.95). This excellent agreement between reciprocal tests is in sharp contrast with the previously reported lack of reciprocity obtained in individual cases (van Regenmortel, 1967; van Regenmortel and von Wechmar, 1970) and it emphasizes again the danger of basing conclusions on too few bleedings from too few animals. The results in Tables 2 and 3 show that a continuous range of increasingly distantly related serotypes of TMV exist and that it
420
M. H. V. VAN REGENMORTEL
would be arbitrary to distinguish between close and distant serological relationships in this case. A distinction between strains and serotypes based on the extent of crossreactivity (Babos and Kassanis, 1963; Kassanis and Phillips, 1970) seems impractical and the present study adds further weight to the suggestion of van Regenmortel and von Wechmar (1970) that the term “serotype” be used to denote any virus strain that is serologically distinguishable from other strains, regardless of whether the serological differences are large or small. The degree of serological relationship between viruses measures indirectly the extent of homology in the amino acid sequence of the corresponding coat proteins. In the case of TMV it is well-known that only a limited number of amino acid residues of the protein contribute to the antigenic determinants of the virus (see van Regenmortel, 1966; 1967). The results in Table 3 show that there is, nevertheless, some correlation between sequence homology and the extent of serological relationship. Furthermore, strain CGMMV-C which has the greatest number of net amino acid exchanges compared to common TMV (30 exchanges) and which appears to have the most divergent sequence (Kurachi et al., 1972) also shows the lowest degree of serological cross-reactivity with TMV. ACKNOWLEDGMENTS The author gratefully acknowledges the skillful assistance of Barbara von Wechmar and Nelly Lelarge. REFERENCES ALLEN, W. R. (1968). Tomato bushy stunt virus from Prunes auium II. Serological typing and the characterization of antibody types and activities. Can.J. Bot. 46, 229-233. BABOS, P., and KASSANIS, B. (1963). Serological relationships and some properties of tobacco necrosis virus strains. J. Gen. Microbial. 32, 135-144. BERCKS, R. (1963). Untersuchungen tiber individuelle Unterschiede von Antiseren gegen Kartoffel X-virus
bei Reaktionen mit verwandten Viren. Phytopathol. z. 47, 301-313. HENNIC B., and W~T~MANN, H. G. (1972). Tobacco mosaic virus = mutants and strains. In “Principles and Techniques in Plant Virology” (C. I. Kado and H. 0. Agrawal, eds.), pp. 546-594. Van Nostrand Reinhold, New York. HOLLINGS, M., and STONE, 0. M. (1965). Studies of pelargonium leaf curl virus II. Relationships to tomato bushy stunt and other viruses. Ann. Appl. Biol. 56, 87-98. KASSANIS, B., and PHILLIPS, M. P. (1970). Serological relationships of strains of tobacco necrosis virus and their ability to activate strains of satellite virus. J. Gen. Viral. 9, 119-126. KOENIG, R., and BERCKS, R. (1968). .&nderungen im heterologen Reaktionsvermogen von Antiseren gegen Vertreter der potato virus X-gruppe im Laufe des Immunisierungsprozesses. Phytopathol. Z. 61, 382-398. KOENIG, R., and GIVORD, L. (1974). Serological interrelationships in the turnip yellow mosaic virus group. virology 58, 119-125. KURACHI, K., FUNATSU, G., FUNATSU, M., and HIDAKA, S. (1972). Partial amino acid sequence of cucumber green mottle mosaic virus coat protein. Agr. Biol. Chem. 36, 1109-1116. Nozu, Y., TOCHIHARA, H., KOMURO, Y., and OKADA, Y. (1971). Chemical and immunological characterization of cucumber green mottle mosaic virus (watermelon strain) protein. Virology 45, 577-585. SENGBUSCH, P. von (1965). Aminoslureaustausche und Tertilrstruktur eines Protein. Vergleich von Mutanten des Tabakmosaikvirus mit serologischen und physikochemischen Methoden. Z. Vererbungslehre 96,364-386. TREMAINE, J. H., and WRIGHT, N. S. (1967). Crossreactive antibodies in antisera to two strains of southern bean mosaic virus. Virology 31, 481-488. VAN R~GENMORTEL, M. H. V. (1966). Plant virus serology. Advan. Virus Res. 12, 207-271. VAN REGENMORTEL,M. H. V. (1967). Serological studies on naturally occurring strains and chemically induced mutants of tobacco mosaic virus. Virology 31, 467-480. VAN REGENMORTEL, M. H. V., and VON WECHMAR, M. B. (1970). A reexamination of the serological relationship between tobacco mosaic virus and cucumber virus 4. Virology 41, 330-338. VAN REGENMORTEL, M. H. V., and VON WECHMAR, M. V. (1974). Serological relationships of strains of tobacco mosaic virus. S. Ajr. J. Sci. 70, 59-60. VON WECHMAR, M. B., and VAN REGENMORTEL, M. H. V. (1970). A simple procedure for purifying tobacco mosaic virus strains. S. Afr. Med. J. 44, 151.
