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Enhanced diffusion of enterovirus antigens in agar gel in the presence of protamine
DISCUSSION
AND
PRELIMINARY
isolate host-range mutants of P221, forming plaques on Ql were unsuccessful (3). Since P221 is a hybrid between P22 and Fels 1 and carries the protein coat of Fels 1, P221 should infect Q1. However, P221 cannot be adsorbed by Ql . These observations suggest that strains lysogenic for Fels 1 may be producing various morphological defectives and variants of this phage, at very high frequencies. This could be analogous to the situation found in E. coli bacteriophage PI (4). With our observations on the artificially damaged genome, it seems likely that spontaneous damage may also be essential for the formation of P221. As we reported previously (3, 5), the genetic homology between P22 and P221 has been measured by recombination experiments. Recently we found that the length of the homologous region between P22 and P221 varied among many independent P221 isolates. Newly isolated P221 groups, P221& and P22ldia, conferred immunity to superinfection with P22 and formed plaques on bacterial strains lysogenic for P221b or P221. Therefore, it is evident that P22 supplies an additional marker Im (immunity), besides c, g and h21, to form P221disl and P221disz. The above finding implies that the length of homology between P22 and P221dis groups is longer than that between P22 and P221 or P221b. From these observations, it may be concluded that P221 arises as a consequence of recombination between P22 and Fels 1 or its defective genome, though no evidence for genetic homology between these phages was noticed. Thus, the genome of P221 consists of parts of P22 and Fels 1. The length of the P22 segment inserted into P221 groups varies from strain to strain of P221 groups. This could be due to the general transducing nature of bacteriophage P22. ACKNOWLEDGMENTS This work was supported in part by grants NIH(AI-06429) and NSF(GB-5773). I wish to thank Dr. E. S. Anderson, England, for supplying 8. typhimurium strain M668. REFERENCES 1. YAMAMOTO, N., and ANDERSON, T. F., Virology 14,430 (1961).
REPORTS
547
2. Y~~.*~o~o,N.,Science 143,144 (1964). 8. YAMAMOTO, N., and WEIR, M. L., Virology 28, 168 (1966). 4. WALKER, D. H., JR., personal communication and Ph.D. Thesis, University of Pennsylvania, 1966. 6. YAMAMOTO, N., and WEIR, M. L., Virology 28, 325 (1966). NOBUTO YAM~MOTO Fels Research Institute and Department of Microbiology Temple University School of Medicine Philadelphia, Pennsylvania 19140 Accepted August .%, 1967
Enhanced Agar
Diffusion Gel
of Enterovirus
in the Presence
Antigens
in
of Protamine’
Lines have been obtained with crude, concentrated preparations of human enteroviruses (poliovirus, Coxsackievirus, echovirus) when they are allowed to react with their corresponding antisera in gel diffusion tests. With Coxsackievirus antigens, two lines were formed in tests with human sera, one of which constituted a (‘group” line, and the other a “specific” line (1). Similar results were obtained in other studies with human sera and echovirus antigens (2). The “group”-specific antigens appear to be associated with capsids free of RNA whereas the “serotype’‘-specific antigens are associated with nucleocapsids (3). In addition, it has been shown that the “group” line is formed by antigen which is not restricted to members of the subgroup, e.g., Coxsackievirus or echovirus, but is apparently distributed throughout the human enteroviruses and represents a major group antigen (2). Although a soluble precursor has been reported for poliovirus capsid (4), soluble antigens have not been demonstrated in human enteroviruses. The lines obtained to date, in gel diffusion tests, would therefore be associated with virions or fragments of virions. Under these conditions, the reactions could be considered to be in the realm of microagglutination (2). The ability of particles to diffuse in agar could thus affect the results obtained in the gel diffusion procedure. 1 This investigation Institutes of Health Fl-GM-18, 899-03.
was supported by National Predoctoral Fellowship, 5-
548
DISCUSSION
AND
PRELIMINARY
Varying results were obtained in gel diffusion tests with human and animal sera using preparations from different strains of the same serotype of enterovirus. Negative or weak reactions, with lines forming close to the antigen well, were observed with echovirus 6 (strains D’Amori and Charles m), echovirus 4 (strain DuToit), and Coxsackievirus B5 (strain N.Y.S. 53122). Infectivity assay, as a criterion for potency of the antigen preparations could not account for the poor results obtained with these particular antigens. In the case of echovirus 6, good reactions were obtained with antigen prepared from strain Charles m+ and in echovirus 4, with prototype strain Pesascek. It was noted that the antigens which yielded weak lines in gel diffusion were prepared from strains which formed minute m type plaques under agar overlay (5). Satisfactory antigens were made from strains which produced large m+ type plaques. Inhibition of viral plaque formation by polyanions present in agar overlay has been reported for a large number of viruses (8). The evidence obtained has suggested that small-plaque formation can be caused by poor diffusion of virus due to electrostatic binding by agar components. The addition of polybasic compounds such as protamine sulfate to the agar reverses the inhibition and allows the formation of larger plaques (5, 7, 8). Thus, by the addition of protamine sulfate to the agar overlay, large mf plaques could be obtained with enterovirus &rains which produced m plaques.
REPORTS
It, seemed reasonable to expect that addition of protamine sulfate to the agar used in gel diffusion might, improve the reactions obtained with antigens of strains producing m plaques. Accordingly, protamine sulfate (Nutritional Biochemical Co.) at a concentration of 1000 pg/ml was incorporated into the 0.8 % Ionagar 30. 2 (Consolidated Laboratories, Inc.) used for gel diffusion studies. The results obtained in the presence and absence of protamine sulfate with echovirus 4 (strain DuToit), and Coxsackievirus B5 (strain N.Y.S. 53122) are shown in Figs. 1 and 2, respectively. Using strain DuToit antigen, no lines of precipitation were observed in the absence of protamine sulfate in the agar (1A) whereas good reactions were obtained in the presence of protamine sulfate (IB). The heavy “group” line appears closer to the antigen well. The “serotype” specific reaction appears as a faint line closer to the serum well (refer 5C). Similarly, strain 53122 formed lines which were very close to the antigen well in the absence of protamine sulfate (2A) and when protamine sulfate was added to the agar, lines appeared at, a distance from the antigen well, were sharply formed and separated from each other (2B). The ‘Lgroup” line is found opposite the well containing serum of an echovirus 4 patient, while the serotype-specific line is observed only opposite wells containing convalescent sera of the Coxsackievirus B5 patient. A similar
effect
of
incorporat’ing
prot,amine
FIG. 1. The effect of protamine on the formation of precipitation lines using echovirus 4 (DuToit) antigen. (A) Agar gel without protamine. (B) Agar gel plus protamine. Cent,ral wells contain DuToit antigen (D). Peripheral wells of both plates contain sera from patients infected with echovirus 4 (Nos. 5, 8, and 9) taken at acute (a) and convalescent (c) phase of illness.
DISCUSSION
AND
PRELIMINARY
549
REPORTS
FIG. 2. The effect of protamine on the formation of precipitation lines using Coxsackievirus B5 (N.Y.S. 53122) antigen. (A) Agar gel without protamine. (B) Agar gel plus protamine. Central wells contain Coxsackievirus B5 antigen (C). Peripheral wells of both plates contain sera from patients infected with Coxsackievirus B5 (Nos. C-f and C-2) taken at acute (a) and convalescent (c) phase of illness. Well 9c contains a convalescent phase serum from a patient infected with echovirus 4.
sulfate in the agar was obtained with echovirus 6, strains D’Amori and Charles m. In contrast, when antigens prepared from strains which produced large m+ plaques were tested under similar conditions, i.e., diffusion in agar gel in the presence and absence of protamine sulfate, no difference in results could be observed, a finding indicative that the protamine sulfate had no effect on the diffusion of these antigens. The improved diffusion of enterovirus antigens in agar gel containing protamine sulfate indicates that the inhibitory mechanism which prevents diffusion of certain enteroviruses in agar and results in m type plaque formation is also operative in gel diffusion tests. This finding should be considered when mutants producing m type plaques are used as the source of antigen. REFERENCES 1. SCHMIDT, N. J., and LENNETTE, E. H., J. Immunol. 89, 85-95 (1962). 2. CONANT, R. M., B.IRRON, A. L., and MILGHOM, F., Proc. Sot. Ezptl. Biol. Med. 121, 307311 (19F6). 3. FORSGREN, M., Acta Palhol. Microbial. Stand. 66, 262-263 (1966). 4. SCH.ZHFF, M. D., MXZEL, J. V., and LEVINTOW, L., Proc. ;\:atl. Acad. Sci. U.S. 51, 329-337 (1964). 6. B‘IRRON, A. L., and KARZON, 1>. T., Am. J. Epidemiol. 81, 323-332 (1965). 6. TAKEMOTO, K. K., Progr. Med. Viral. 8, 314-348 (1966). 7. LIEBH.\BER, H., and T.ZKEMOTO, K. K., Virology 14, 502-504 (1961).
8. TYTELL, A. A., and NEUMAN, R. E., Proc. Sot. Exptl. Biol. Med. 113, 343-346 (1963). ROBERT M. CONANT~ ALMEN L. B.IRRON Department of Bacteriology and Immunology School of Medicine State University of New York at Bu$alo Bu$alo, New York Accepted September 6, 1967 2 Present address: Departments of Pediatrics and Medical Microbiology, Ohio Stat,e University, Children’s Hospital, Columbus, Ohio 43205
Aphid
Transmission Correlated
with
of Virus
from
intracellular
Leaf
Sectors
Inclusions’
Numerous studies have been made in which various leaves from the same plant were compared as virus sources for aphids; proportionately fewer studies have been done, however, to determine the availability of virus throughout individual leaves (1, 2). Using a virus that caused systemically infected leaves of Crotalaria spectabilis Roth to sector into clearly discernible areas of light and dark green (Fig. I), we were able (a) to determine the greater infectivity of the epidermis of light green sectors and (b) to correlate this observation wit’h the occurrence of intracellular inclusions in epidermal cells from these sectors. Bradley (3) reported that the epidermis of light green areas from leaves exhibiting 1 Florida Agricultural Journal Series, No. 2722.
Experiment
Stations
AND
PRELIMINARY
isolate host-range mutants of P221, forming plaques on Ql were unsuccessful (3). Since P221 is a hybrid between P22 and Fels 1 and carries the protein coat of Fels 1, P221 should infect Q1. However, P221 cannot be adsorbed by Ql . These observations suggest that strains lysogenic for Fels 1 may be producing various morphological defectives and variants of this phage, at very high frequencies. This could be analogous to the situation found in E. coli bacteriophage PI (4). With our observations on the artificially damaged genome, it seems likely that spontaneous damage may also be essential for the formation of P221. As we reported previously (3, 5), the genetic homology between P22 and P221 has been measured by recombination experiments. Recently we found that the length of the homologous region between P22 and P221 varied among many independent P221 isolates. Newly isolated P221 groups, P221& and P22ldia, conferred immunity to superinfection with P22 and formed plaques on bacterial strains lysogenic for P221b or P221. Therefore, it is evident that P22 supplies an additional marker Im (immunity), besides c, g and h21, to form P221disl and P221disz. The above finding implies that the length of homology between P22 and P221dis groups is longer than that between P22 and P221 or P221b. From these observations, it may be concluded that P221 arises as a consequence of recombination between P22 and Fels 1 or its defective genome, though no evidence for genetic homology between these phages was noticed. Thus, the genome of P221 consists of parts of P22 and Fels 1. The length of the P22 segment inserted into P221 groups varies from strain to strain of P221 groups. This could be due to the general transducing nature of bacteriophage P22. ACKNOWLEDGMENTS This work was supported in part by grants NIH(AI-06429) and NSF(GB-5773). I wish to thank Dr. E. S. Anderson, England, for supplying 8. typhimurium strain M668. REFERENCES 1. YAMAMOTO, N., and ANDERSON, T. F., Virology 14,430 (1961).
REPORTS
547
2. Y~~.*~o~o,N.,Science 143,144 (1964). 8. YAMAMOTO, N., and WEIR, M. L., Virology 28, 168 (1966). 4. WALKER, D. H., JR., personal communication and Ph.D. Thesis, University of Pennsylvania, 1966. 6. YAMAMOTO, N., and WEIR, M. L., Virology 28, 325 (1966). NOBUTO YAM~MOTO Fels Research Institute and Department of Microbiology Temple University School of Medicine Philadelphia, Pennsylvania 19140 Accepted August .%, 1967
Enhanced Agar
Diffusion Gel
of Enterovirus
in the Presence
Antigens
in
of Protamine’
Lines have been obtained with crude, concentrated preparations of human enteroviruses (poliovirus, Coxsackievirus, echovirus) when they are allowed to react with their corresponding antisera in gel diffusion tests. With Coxsackievirus antigens, two lines were formed in tests with human sera, one of which constituted a (‘group” line, and the other a “specific” line (1). Similar results were obtained in other studies with human sera and echovirus antigens (2). The “group”-specific antigens appear to be associated with capsids free of RNA whereas the “serotype’‘-specific antigens are associated with nucleocapsids (3). In addition, it has been shown that the “group” line is formed by antigen which is not restricted to members of the subgroup, e.g., Coxsackievirus or echovirus, but is apparently distributed throughout the human enteroviruses and represents a major group antigen (2). Although a soluble precursor has been reported for poliovirus capsid (4), soluble antigens have not been demonstrated in human enteroviruses. The lines obtained to date, in gel diffusion tests, would therefore be associated with virions or fragments of virions. Under these conditions, the reactions could be considered to be in the realm of microagglutination (2). The ability of particles to diffuse in agar could thus affect the results obtained in the gel diffusion procedure. 1 This investigation Institutes of Health Fl-GM-18, 899-03.
was supported by National Predoctoral Fellowship, 5-
548
DISCUSSION
AND
PRELIMINARY
Varying results were obtained in gel diffusion tests with human and animal sera using preparations from different strains of the same serotype of enterovirus. Negative or weak reactions, with lines forming close to the antigen well, were observed with echovirus 6 (strains D’Amori and Charles m), echovirus 4 (strain DuToit), and Coxsackievirus B5 (strain N.Y.S. 53122). Infectivity assay, as a criterion for potency of the antigen preparations could not account for the poor results obtained with these particular antigens. In the case of echovirus 6, good reactions were obtained with antigen prepared from strain Charles m+ and in echovirus 4, with prototype strain Pesascek. It was noted that the antigens which yielded weak lines in gel diffusion were prepared from strains which formed minute m type plaques under agar overlay (5). Satisfactory antigens were made from strains which produced large m+ type plaques. Inhibition of viral plaque formation by polyanions present in agar overlay has been reported for a large number of viruses (8). The evidence obtained has suggested that small-plaque formation can be caused by poor diffusion of virus due to electrostatic binding by agar components. The addition of polybasic compounds such as protamine sulfate to the agar reverses the inhibition and allows the formation of larger plaques (5, 7, 8). Thus, by the addition of protamine sulfate to the agar overlay, large mf plaques could be obtained with enterovirus &rains which produced m plaques.
REPORTS
It, seemed reasonable to expect that addition of protamine sulfate to the agar used in gel diffusion might, improve the reactions obtained with antigens of strains producing m plaques. Accordingly, protamine sulfate (Nutritional Biochemical Co.) at a concentration of 1000 pg/ml was incorporated into the 0.8 % Ionagar 30. 2 (Consolidated Laboratories, Inc.) used for gel diffusion studies. The results obtained in the presence and absence of protamine sulfate with echovirus 4 (strain DuToit), and Coxsackievirus B5 (strain N.Y.S. 53122) are shown in Figs. 1 and 2, respectively. Using strain DuToit antigen, no lines of precipitation were observed in the absence of protamine sulfate in the agar (1A) whereas good reactions were obtained in the presence of protamine sulfate (IB). The heavy “group” line appears closer to the antigen well. The “serotype” specific reaction appears as a faint line closer to the serum well (refer 5C). Similarly, strain 53122 formed lines which were very close to the antigen well in the absence of protamine sulfate (2A) and when protamine sulfate was added to the agar, lines appeared at, a distance from the antigen well, were sharply formed and separated from each other (2B). The ‘Lgroup” line is found opposite the well containing serum of an echovirus 4 patient, while the serotype-specific line is observed only opposite wells containing convalescent sera of the Coxsackievirus B5 patient. A similar
effect
of
incorporat’ing
prot,amine
FIG. 1. The effect of protamine on the formation of precipitation lines using echovirus 4 (DuToit) antigen. (A) Agar gel without protamine. (B) Agar gel plus protamine. Cent,ral wells contain DuToit antigen (D). Peripheral wells of both plates contain sera from patients infected with echovirus 4 (Nos. 5, 8, and 9) taken at acute (a) and convalescent (c) phase of illness.
DISCUSSION
AND
PRELIMINARY
549
REPORTS
FIG. 2. The effect of protamine on the formation of precipitation lines using Coxsackievirus B5 (N.Y.S. 53122) antigen. (A) Agar gel without protamine. (B) Agar gel plus protamine. Central wells contain Coxsackievirus B5 antigen (C). Peripheral wells of both plates contain sera from patients infected with Coxsackievirus B5 (Nos. C-f and C-2) taken at acute (a) and convalescent (c) phase of illness. Well 9c contains a convalescent phase serum from a patient infected with echovirus 4.
sulfate in the agar was obtained with echovirus 6, strains D’Amori and Charles m. In contrast, when antigens prepared from strains which produced large m+ plaques were tested under similar conditions, i.e., diffusion in agar gel in the presence and absence of protamine sulfate, no difference in results could be observed, a finding indicative that the protamine sulfate had no effect on the diffusion of these antigens. The improved diffusion of enterovirus antigens in agar gel containing protamine sulfate indicates that the inhibitory mechanism which prevents diffusion of certain enteroviruses in agar and results in m type plaque formation is also operative in gel diffusion tests. This finding should be considered when mutants producing m type plaques are used as the source of antigen. REFERENCES 1. SCHMIDT, N. J., and LENNETTE, E. H., J. Immunol. 89, 85-95 (1962). 2. CONANT, R. M., B.IRRON, A. L., and MILGHOM, F., Proc. Sot. Ezptl. Biol. Med. 121, 307311 (19F6). 3. FORSGREN, M., Acta Palhol. Microbial. Stand. 66, 262-263 (1966). 4. SCH.ZHFF, M. D., MXZEL, J. V., and LEVINTOW, L., Proc. ;\:atl. Acad. Sci. U.S. 51, 329-337 (1964). 6. B‘IRRON, A. L., and KARZON, 1>. T., Am. J. Epidemiol. 81, 323-332 (1965). 6. TAKEMOTO, K. K., Progr. Med. Viral. 8, 314-348 (1966). 7. LIEBH.\BER, H., and T.ZKEMOTO, K. K., Virology 14, 502-504 (1961).
8. TYTELL, A. A., and NEUMAN, R. E., Proc. Sot. Exptl. Biol. Med. 113, 343-346 (1963). ROBERT M. CONANT~ ALMEN L. B.IRRON Department of Bacteriology and Immunology School of Medicine State University of New York at Bu$alo Bu$alo, New York Accepted September 6, 1967 2 Present address: Departments of Pediatrics and Medical Microbiology, Ohio Stat,e University, Children’s Hospital, Columbus, Ohio 43205
Aphid
Transmission Correlated
with
of Virus
from
intracellular
Leaf
Sectors
Inclusions’
Numerous studies have been made in which various leaves from the same plant were compared as virus sources for aphids; proportionately fewer studies have been done, however, to determine the availability of virus throughout individual leaves (1, 2). Using a virus that caused systemically infected leaves of Crotalaria spectabilis Roth to sector into clearly discernible areas of light and dark green (Fig. I), we were able (a) to determine the greater infectivity of the epidermis of light green sectors and (b) to correlate this observation wit’h the occurrence of intracellular inclusions in epidermal cells from these sectors. Bradley (3) reported that the epidermis of light green areas from leaves exhibiting 1 Florida Agricultural Journal Series, No. 2722.
Experiment
Stations