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The serological relationship between tobacco mosaic virus and cucumber viruses 3 and 4
174
SHORT
COillMU~ICATIOiYS
The Serological Relationship between Tobacco Mosaic Virus and Cucumber Viruses 3 and 4 Bawden and Pirie (1) in 1937 showed that cucumber viruses 3 and 4 (CV 3 and 4) were serologically related to tobacco mosaic virus (TMV), but less closely than tomato aucuba mosaic virus (TAMV) and tomato enation mosaic virus (TEMV), which were previously recognised as strains of TMV. They could demonstrate serological differences between TAMV or TEMV and TMV only bJ test,s with antisera that had been absorbed with heterologous antigens, whereas differences between CV 3 or 4 and the strains that infect solanaceous plants were evident from simple precipitation tests. Thus, with its homologous antiserum at a const’ant dilution of 1: 50, each virus precipitated rapidly and the optimal precipitation point was with virus at 1 mg or more per milliliter. By contrast, with TMV and antiserum to CV 3 or 4, or CV 3 or 4 with antiserum to T&IV, TAMV, or TEMV, precipitation was slow, t’here were large zones in which antigen excess inhibited precipitation, and the optimal precipitation was with an antiserum: virus ratio more than 100 times larger than with homologous antiserum. Since 1937 many other viruses have been found that show similar remote serological relationships to each other as do CV 3 or 4 to TMV. In addit,ion to giving zones in which precipitation is inhibited by antigen excess, antisera to such viruses give much smaller precipitation end points when titrated against heterologous than against homologous virus, and their titres with the homologous virus remain almost unchanged after absorption wit,h the heterologous virus. Viruses that are serologically related but produce antisera containing few common a&bodies usually also differ from one another in many other ways, not only in host, range but, also in physical properties and in their amino acid composition. Thus, Knight (2) considered t,hat CV 3 and 4 differed so much from TJIV t,hat, he was inclined to neglect t,he serological relat,ionship and to conclude t,hat they should be regarded as distinct from TJIV. However, all the viruses that have been found to have common antigens nlso share man\- ot,her proper-
ties, especially gross morphological characters, and there seems to be no better way of grouping viruses than by serological relatedness. Useful subdivisions within the groups can also be made depending on how similar or dissimilar are their ant,isera, and Kassanis (S) suggested that the term strain should be restricted to viruses closely related serologitally and the term serotype applied to those readily distinguished from one another serologically. The distinction between serotype and strain can be expressed quantitatively by the difference between the t)itre of’ the sera when titrated against homologous and heterologous virus or by the amount the titre with the homologous virus is changed b!. absorption wi-ith the heterologous virus. Thus, \vit’h serotypes, titres of heterologous sera are only a small fraction of those lvith homologous sera, whereas with strains, titres of homologous and heterologous sera differ little. Also, absorption with a serotype decreases t)he titre very little, whereas absorpt,ion with a strain removes almost, all the antibodies. Hence, TAl\IV and TEJIV are classed as strains of T;\IV, but CV 3 and 4 as serotypes. van Regenmort,el (J), from work with CV 8 and 4 and various strains of TJIV, has challenged the value of serological relatedness in indicating closeness of similarities between viruses. Although working in the same labomtory as Knight and presumably with t’he same virus stocks, he has reached an opposite conclusion and stated that, from their behaviour with antiserum to CV 4, TJIV and CV 4 “both seem to qualify as closely relat’ed strains.” He st’at#ed that, Ivhether the t,wo were rated as serotypes or strains depended on whether t’hey mere test)ed with antiserum to TJlV or to CV 4 and PO discredits “the distinction between close and distant serological relatSionship as well as thalt between strains and serotypes.” Hojvever, his experiment:d result,s arc in such conflict, not only with those of l%~~vtlen and Pirie (1). hut :dso with those w h:\~ obtained using a strain recently isoMet1 t’rom a tlaturally infected cucumber plant, that’ his conclusion cannot go un~hallcnged. We have compared T1lV with this strait1 (C’V), \vhich closel\~ resembles (11’ 1, ant1 \vit h Ihcl hcan form of tobacco mosaic virus (R.\lV). \vhich
SHORT
infects beans systemically (5). Table 1 shows that the titres of antisera to all three viruses are very much smaller against heterologous than against homologous antigens and absorption with the heterologous leaves the titres with the homologous unchanged. Rabbits are individuals, and different ones give sera of different potency when injected with the same amount of one antigen. Hence different rabbits can also be expected to respond differently to the major and minor antigens of a virus, and exact reciprocity is not to be expected between tests with serotypes, but whichever way around our tests were made we always found the same order of differences. van Regenmortel’s results with TMV antisera, a titre of l/2048 with TMV and one of l/32 with CV 4, differ from ours (l/1024 and l/32) by a factor of 2, which is the sort of variation we encounter, but his results with CV 4 antiserum, a titre of only l/512 with CV 4 yet l/128 with TMV, not only differ from anything we have TABLE
I
THE RECIPROCALS OF PRECIPITATION END POINTS OF ABSORBED AND UNABSORBED ANTISERA WITH HOMOLOGOUS AND HETEROLOGOUS ANTIGENS
Antiserum
TMV TMV TMV BMV BMV BMV cv cv
Antigen used for absorption BMV CV cv TMV TMV -
Antigen used in precipitation tests TMV 1024 1024 1024 16 0 16 0 16
175
COMMUNICATIONS
BMV
CV
0 0 16 512 512 512 0 0
8 0 32 0 2 4 5120 5120
o The antisera were obtained by injecting rabbits at weekly intervals with 5 mg of virus. Three injections were given with TMV and BMV and five with CT:. The rabbits were bled 10 days after the last injection. For absorption, antisera were mixed wit.h an excess of the specific antigen and the precipitate was removed by centrifugation. The supernatant fluid was used in precipitation tests; 0.4 ml containing 4 pg of antigen was added to 0.4 ml of serum at various dilutions, and the end point was taken as the greatest dilution at which there was a visible precipitate after 10 hours’ incubation at 50”.
found with serotypes of TMV but also with serotypes of other viruses. His results seem inexplicable by differences between individual rabbits because he states that 2 to 4 rabbits were injected with each antigen. The lack of reciprocity we have experienced is not such that the behaviour with one antiserum suggests the distant relationship of serotype and with another the close relationship of strain, but that one antiserum may suggest serotype relationship and another no relationship. This shows in Table 1 by the failure of CV antiserum to precipitate BMV, although both other antisera precipitated all three viruses. The lack of reciprocity is in the failure of the quantitatively minor antibodies to become plentiful enough to be detectable rather than to become abundant. However, this result also has some interest in suggesting that the virus from cucumber, although only remotely related serologically to TMV, may be nearer to it in some structural manner than it is to BMV. This suggestion gets some support from the results of tests with absorbed sera, for whereas absorbing TMV antiserum with CV removed all antibodies to BMV, absorbing with BI\/IV Ieft some able to precipitate CV. Until the identity of virus antigens is established, the taxonomic significance of serological relatedness will remain in debate, but meanwhile the value of serology, not only as a simple and quick method of grouping viruses, but also for indicating their order of similarities, is too great to be lightly dismissed. REFERENCES 1. BAWDEN, F. C., and PIRIE, N. W., Rrit. J. Exptl. Pathol. 18, 275-291 (1937). 2. KNIGHT, C. A., J’ii’rology 1,261-267 (1955). 3. KASSANIS, B., European Potato J. 4, 13-24 (1961). 4. v.4~ REGENMDRTEL, M. H. V., vi’irology 31, 467480 (1967). 5. B~WDEN. F. C., J. Gen. Microbial. 18, 751-766 (1958). F. C. BAWDEN B. Kass.4~1~ Rothamsted Experimental Station Harpenden, Hertfordshire England Accepted October 20, 1967
SHORT
COillMU~ICATIOiYS
The Serological Relationship between Tobacco Mosaic Virus and Cucumber Viruses 3 and 4 Bawden and Pirie (1) in 1937 showed that cucumber viruses 3 and 4 (CV 3 and 4) were serologically related to tobacco mosaic virus (TMV), but less closely than tomato aucuba mosaic virus (TAMV) and tomato enation mosaic virus (TEMV), which were previously recognised as strains of TMV. They could demonstrate serological differences between TAMV or TEMV and TMV only bJ test,s with antisera that had been absorbed with heterologous antigens, whereas differences between CV 3 or 4 and the strains that infect solanaceous plants were evident from simple precipitation tests. Thus, with its homologous antiserum at a const’ant dilution of 1: 50, each virus precipitated rapidly and the optimal precipitation point was with virus at 1 mg or more per milliliter. By contrast, with TMV and antiserum to CV 3 or 4, or CV 3 or 4 with antiserum to T&IV, TAMV, or TEMV, precipitation was slow, t’here were large zones in which antigen excess inhibited precipitation, and the optimal precipitation was with an antiserum: virus ratio more than 100 times larger than with homologous antiserum. Since 1937 many other viruses have been found that show similar remote serological relationships to each other as do CV 3 or 4 to TMV. In addit,ion to giving zones in which precipitation is inhibited by antigen excess, antisera to such viruses give much smaller precipitation end points when titrated against heterologous than against homologous virus, and their titres with the homologous virus remain almost unchanged after absorption wit,h the heterologous virus. Viruses that are serologically related but produce antisera containing few common a&bodies usually also differ from one another in many other ways, not only in host, range but, also in physical properties and in their amino acid composition. Thus, Knight (2) considered t,hat CV 3 and 4 differed so much from TJIV t,hat, he was inclined to neglect t,he serological relat,ionship and to conclude t,hat they should be regarded as distinct from TJIV. However, all the viruses that have been found to have common antigens nlso share man\- ot,her proper-
ties, especially gross morphological characters, and there seems to be no better way of grouping viruses than by serological relatedness. Useful subdivisions within the groups can also be made depending on how similar or dissimilar are their ant,isera, and Kassanis (S) suggested that the term strain should be restricted to viruses closely related serologitally and the term serotype applied to those readily distinguished from one another serologically. The distinction between serotype and strain can be expressed quantitatively by the difference between the t)itre of’ the sera when titrated against homologous and heterologous virus or by the amount the titre with the homologous virus is changed b!. absorption wi-ith the heterologous virus. Thus, \vit’h serotypes, titres of heterologous sera are only a small fraction of those lvith homologous sera, whereas with strains, titres of homologous and heterologous sera differ little. Also, absorption with a serotype decreases t)he titre very little, whereas absorpt,ion with a strain removes almost, all the antibodies. Hence, TAl\IV and TEJIV are classed as strains of T;\IV, but CV 3 and 4 as serotypes. van Regenmort,el (J), from work with CV 8 and 4 and various strains of TJIV, has challenged the value of serological relatedness in indicating closeness of similarities between viruses. Although working in the same labomtory as Knight and presumably with t’he same virus stocks, he has reached an opposite conclusion and stated that, from their behaviour with antiserum to CV 4, TJIV and CV 4 “both seem to qualify as closely relat’ed strains.” He st’at#ed that, Ivhether the t,wo were rated as serotypes or strains depended on whether t’hey mere test)ed with antiserum to TJlV or to CV 4 and PO discredits “the distinction between close and distant serological relatSionship as well as thalt between strains and serotypes.” Hojvever, his experiment:d result,s arc in such conflict, not only with those of l%~~vtlen and Pirie (1). hut :dso with those w h:\~ obtained using a strain recently isoMet1 t’rom a tlaturally infected cucumber plant, that’ his conclusion cannot go un~hallcnged. We have compared T1lV with this strait1 (C’V), \vhich closel\~ resembles (11’ 1, ant1 \vit h Ihcl hcan form of tobacco mosaic virus (R.\lV). \vhich
SHORT
infects beans systemically (5). Table 1 shows that the titres of antisera to all three viruses are very much smaller against heterologous than against homologous antigens and absorption with the heterologous leaves the titres with the homologous unchanged. Rabbits are individuals, and different ones give sera of different potency when injected with the same amount of one antigen. Hence different rabbits can also be expected to respond differently to the major and minor antigens of a virus, and exact reciprocity is not to be expected between tests with serotypes, but whichever way around our tests were made we always found the same order of differences. van Regenmortel’s results with TMV antisera, a titre of l/2048 with TMV and one of l/32 with CV 4, differ from ours (l/1024 and l/32) by a factor of 2, which is the sort of variation we encounter, but his results with CV 4 antiserum, a titre of only l/512 with CV 4 yet l/128 with TMV, not only differ from anything we have TABLE
I
THE RECIPROCALS OF PRECIPITATION END POINTS OF ABSORBED AND UNABSORBED ANTISERA WITH HOMOLOGOUS AND HETEROLOGOUS ANTIGENS
Antiserum
TMV TMV TMV BMV BMV BMV cv cv
Antigen used for absorption BMV CV cv TMV TMV -
Antigen used in precipitation tests TMV 1024 1024 1024 16 0 16 0 16
175
COMMUNICATIONS
BMV
CV
0 0 16 512 512 512 0 0
8 0 32 0 2 4 5120 5120
o The antisera were obtained by injecting rabbits at weekly intervals with 5 mg of virus. Three injections were given with TMV and BMV and five with CT:. The rabbits were bled 10 days after the last injection. For absorption, antisera were mixed wit.h an excess of the specific antigen and the precipitate was removed by centrifugation. The supernatant fluid was used in precipitation tests; 0.4 ml containing 4 pg of antigen was added to 0.4 ml of serum at various dilutions, and the end point was taken as the greatest dilution at which there was a visible precipitate after 10 hours’ incubation at 50”.
found with serotypes of TMV but also with serotypes of other viruses. His results seem inexplicable by differences between individual rabbits because he states that 2 to 4 rabbits were injected with each antigen. The lack of reciprocity we have experienced is not such that the behaviour with one antiserum suggests the distant relationship of serotype and with another the close relationship of strain, but that one antiserum may suggest serotype relationship and another no relationship. This shows in Table 1 by the failure of CV antiserum to precipitate BMV, although both other antisera precipitated all three viruses. The lack of reciprocity is in the failure of the quantitatively minor antibodies to become plentiful enough to be detectable rather than to become abundant. However, this result also has some interest in suggesting that the virus from cucumber, although only remotely related serologically to TMV, may be nearer to it in some structural manner than it is to BMV. This suggestion gets some support from the results of tests with absorbed sera, for whereas absorbing TMV antiserum with CV removed all antibodies to BMV, absorbing with BI\/IV Ieft some able to precipitate CV. Until the identity of virus antigens is established, the taxonomic significance of serological relatedness will remain in debate, but meanwhile the value of serology, not only as a simple and quick method of grouping viruses, but also for indicating their order of similarities, is too great to be lightly dismissed. REFERENCES 1. BAWDEN, F. C., and PIRIE, N. W., Rrit. J. Exptl. Pathol. 18, 275-291 (1937). 2. KNIGHT, C. A., J’ii’rology 1,261-267 (1955). 3. KASSANIS, B., European Potato J. 4, 13-24 (1961). 4. v.4~ REGENMDRTEL, M. H. V., vi’irology 31, 467480 (1967). 5. B~WDEN. F. C., J. Gen. Microbial. 18, 751-766 (1958). F. C. BAWDEN B. Kass.4~1~ Rothamsted Experimental Station Harpenden, Hertfordshire England Accepted October 20, 1967