virus is inactivated by such dilutions (Afanasiev and Morris, 1952; Kim and Hagendorn, 1959; Patino and Zaumeyer, 1959; Smith, 1957;

Transcription

1 STABILITY OF TOBACCO-MOSAIC VIRUS, MARMOR TABACI H, IN SOLUTIONS DILUTED BEYOND THE END POINT OF INFECTIVITY H. H. THORNBERRY AND B. B. NAGAICH' Department of Plant Pathology, University of Illinois, Urbana, Illinois Received for publication January 2, 1962 ABSTRACT THORNBERRY, H. H. (University of Illinois, Urbana, Ill.) AND B. B. NAGAICH. Stability of tobacco-mosaic virus, Marmor tabaci H, in solutions diluted beyond the end point of infectivity. J. Bacteriol. 83: Tobacco-mosaic virus (TMV) in crude plant extract and in a partially purified preparation was diluted in distilled water, 0.1 M potassium phosphate buffer, and 0.1 M sodium chloride beyond detectable infectivity by usual assays on 12 primary leaves of Scotia beans, Phaseolus vulgaris L. All assays were made with inoculum containing abrasive at ph 8.5 in 0.1 M phosphate buffer. Infectious virus was recovered from each highly diluted solution (10 liters at ph 7.5 and 4 C for 24 hr) by adsorption at ph 2.5 on Celite particles and desorption at ph 8.5 and 40 C with 100 or 150 ml of 0.1 M phosphate buffer. Thus, TMV is not irreversibly inactivated by such dilutions. To ascertain the infectivity of the virus at high dilutions, TMV in crude plant extracts at three dilutions (10-6, 10-7, and 10-8) was assayed on bean leaves (12 leaves for 10-6, 120 leaves for 10-7, and 1,200 leaves for 10-8). Infection was obtained from each diluted inoculum (10-6, 26 total local lesions; 107, 30; and 10-8, 22). These data support the conclusions that TMV remains infectious at high dilutions and that the failure to obtain infection beyond the end point of infectivity by usual assays is owing to sparsity of infectious virus rather than to viral inactivation. In addition, they suggest that a single viral particle is capable of infecting a susceptible site. Titer of virus in expressed juice from diseased plants is an important characteristic of viruses, and is generally expressed as the dilution end point of infectivity. Infectiousness of virus in 1 Graduate student on Graduate College Fellowship for Ph.D. Degree. Present address: Flowerdale, Simla-E, India. solutions diluted beyond the end point of infectivity has not been determined, yet the literature contains statements to the effect that virus is inactivated by such dilutions (Afanasiev and Morris, 1952; Kim and Hagendorn, 1959; Patino and Zaumeyer, 1959; Smith, 1957; Thomas, 1951; Zaumeyer and Thomas, 1950; Zaumeyer and Patino, 1960). This paper reports the results of experiments designed to ascertain the stability of tobacco-mosaic virus (TMV) at dilutions beyond which infectivity is not detectable by the usual plant bio-assay. The hypothesis advanced to guide the investigations contended that the virus is not inactivated by high dilutions, but that the infectious particles or units in high dilutions become too sparse for detection by the bioassays employed. The findings affirm this hypothesis and give substantial support to the conclusion that TMV is infectious at high dilutions. MATERIALS AND METHODS Virus. The TMV used was a subculture of Marmor tabaci H (ATCC no. 2), which was increased in plants of Turkish tobacco, Nicotiana tabacum L. The samples of crude extract were prepared from infected leaves by crushing the frozen tissues with a hand-operated food grinder and straining the liquid through 4-ply cheesecloth. The partially purified virus was prepared from the crude juice by two cycles of differential centrifugation. The clarification runs were at 10,000 X g (average force times gravity) for 10 min; the first sedimentation was run at 40,000 X g for 4 hr, the second at 80,000 X g for 2 hr. The preparation contained 2.8 mg protein per ml, determined by micro-kjeldahl nitrogen analysis of protein precipitated by 10% trichloroacetic acid at 70 C. Filtered samples of the crude juice were obtained by filtering a 10-2 dilution of the juice in 0.1 M potassium phosphate buffer at ph 8.5 through a sterile bacteriological candle (Mandler 9 lb) under 635 mm Hg negative pressure. 1322

2 19621 STABILITY OF TOBACCO-MOSAIC VIRUS 1323 Bio-assay. The bio-assays for virus consisted of inoculating the upper surface of primary leaves of Scotia beans, Phaseolus vulgaris L. Inoculations were made on the 12th to 14th day after the planting of seed in sterilized soil in sterilized 4-in. pots in a greenhouse at 25 5 C. The inoculum was at optimal conditions for maximal infection, i.e., ph 8.5 and 0.1 M phosphate buffer (Thornberry, 1935b) and containing 7% abrasive, 800-mesh aluminum oxide powder (Beraha, Varzandeh, and Thornberry, 1955). Inoculations were made by stroking the leaves with a cheesecloth pad moistened with the inoculum. Immediately before inoculation, the inoculum was agitated to suspend the abrasive; within 1 min after inoculation, the rubbed leaves were washed with a spray of tap water. Necrotic local lesions were counted 1 week after the test plants were inoculated. Dilutions and diluents. Viral dilutions were made in tenfold steps in three diluents: distilled water, 0.1 M potassium phosphate solution, and 0.1 M sodium chloride solution. When necessary, the H-ion concentration was adjusted to ph 7.5 with 1 N HCl or KOH. The 10 liters of diluted viral solutions were stored for 24 hr at 4 C to permit equilibration of the solutions. Concentration of virus. The highly diluted solutions were concentrated by adsorption of virus to Celite particles at ph 2.5 and 4 C and by desorption from the Celite at ph 8.5 and 40 C. The 10 liters of viral solutions were adjusted to ph 2.5 and filtered through a filter cake of compacted Celite particles. To prepare the filter cake, a slurry of 25 g of air-dried Celite powder (Johns-Manville no. 209 "Hy-Flow") in about 1 liter of distilled water was filtered through paper (150 mm circles of Whatman no. 1 in a Biichner funnel with about 635 mm of Hg negative pressure). The Celite particles that lodged on the filter paper were firmly packed by pounding with the flat end of a pestle. Compactness of the cake was important to assure the retention of the virus particles by the filter cake. For desorption, the filter cake with adsorbed or lodged virus was suspended in 100 or 150 ml of 0.1 M phosphate buffer at ph 8.5 and heated to 40 C. After thorough agitation, the slurry was filtered through filter paper under 635 mm of Hg negative pressure. The filtrate was refiltered twice through the noncompacted cake of Celite to assure maximal desorption. RESULTS With present techniques applicable to virus studies, it should be possible to concentrate infectious virus from highly diluted solutions to the point where the virus can be detected by usual assays. To test this reasoning, TMV at high dilutions in 10 liters of diluents was adsorbed to Celite particles and desorbed with phosphate buffer as described above. TMV in a purified preparation is not irreversibly inactivated when diluted to 10-7 in distilled water or a 0.1 M solution of NaCl (Table 1). Solutions containing the viral preparation at TABLE 1. Infectiousness of partially purified tobacco-mosaic virus at varied dilutions and in fractions concentratedfrom solutions diluted beyond the end point of infectivity Bioassay Virus diluted in Samples Dilution Distilled water* 0.1 M NaCl* Purified preparation Purified preparation i 2 Purified preparation Purified preparation Purified preparation at 1)7, 10 liters at ph 7.5 None 0 0 for 24 hr Adsorptive 10-7 filtrate, 10 liters, ph 2.5 None 0 0 Eluate,t 150 ml None i 10 Eluate,t 150 ml i Eluate,t 150 ml i Eluate,t 150 ml * Mean of the total number of necrotic local lesions on 28 bean leaves in two experiments; the second figure gives the standard error of the mean. t Eluate was the Celite washing with 150 ml of 0.1 M potassium phosphate buffer at ph 8.5.

3 1324 THORNBERRY AND NAGAICH [vol. 83 TABLE 2. Infectiousness of tobacco-mosaic virus in crude plant extract at varied dilutions and in fractions concentrated from solutions diluted beyond the end point of infectivity Bio-assay Virus diluted in 0.1 m 0.1 MP04 Buffer Samples Dilution Distilled water NaCi Unfiltered Filtered Crude extract * Crude extract Crude extract 10-v Crude extract at 10-7, 10 liters at ph None for 24 hr Adsorptive 10-7 filtrate, 10 liters, ph None Eluate,t 100 ml None Eluate,t 100 ml Eluate,t 100 ml Eluate,t 100 ml Resuspended Celite, 100 ml None Resuspended Celite, 100 ml * Mean of the total number of necrotic local lesions on 12 bean leaves in one experiment. (Assays on the crude extract were done on the day previous to other assays.) t Eluate was the Celite washing with 100 ml of 0.1 M potassium phosphate buffer at ph 8.5. a dilution of 10- were noninfectious as measured by the bio-assay test involving 14 bean leaves per test. TMV in crude extract is likewise not irreversibly inactivated by a 10-7dilution in distilled water, 0.1 M NaCl, or 0.1 M phosphate buffer (Table 2). Filtration of the crude juice through a bacteriological candle did not alter the stability of the virus to dilution. The susceptibility of the group of plants used in this experiment for assays on diluted crude juice was apparently greater than usual groups of plants, because a few infections occurred from the 10-7 dilutions which generally give no lesions in assays. These samples of diluted crude juice were assayed 1 day prior to the other assays. No infection was obtained from samples of the 10-7 dilutions that had been in storage for 24 hr. From the data in Tables 1 and 2, one may conclude that the virus is not irreversibly inactivated by dilution. They do not indicate, however, the infectiousness of the virus while highly diluted. To ascertain the infectiousness of the virus at high dilutions, TMV in crude juice was diluted to 10-6, 10-7, and 10- in 0.1 M phosphate buffer and assayed on 12 bean leaves with the 10-1 inoculum, 120 leaves with the 10-7 inoculum, and 1,200 leaves with the 10-8 inoculum. The results from these tests were: 26 necrotic local lesions from the 10-6 dilution, 30 lesions from the 10-7 dilution, and 22 lesions from the 10-8 dilution. Thus, the virus is obviously infectious while it is highly diluted. DISCUSSION The influence of electrolytes on the infectivity of TMV at high dilutions was studied because the ionic environment might influence the stability of the virus upon dilution. Stanley (1935, 1936) reported that the isolated TMV protein had globulin-like properties (globulins being soluble in aqueous solutions of neutral electrolytes, but insoluble in distilled water). In some of the earlier experiments, active virus was not recovered from the diluted solutions. These negative results suggested that the virus was either inactivated by the high dilutions oi was not sufficiently concentrated by adsorption and desorption for detection. In checking the supply of distilled water used in these experiments, it was found that the water was impure because of faulty distilling equipment. Apparently, salts in the dilution medium interfered with adsorption or desorption to the extent that an insufficient amount of virus was recovered for detection by the assays. Since active virus was recovered in subsequent experiments in which pure water was used for diluents, it was assured that a-concentration of 0.1 M NaCl or phosphate

4 19621 STABILITY OF TOBACCO-MOSAIC VIRUS 1325 buffer had no permanent influence on the infectivity of TMV at the dilutions tested. The effect of filtering TMV through a bacteriological candle might influence the infectivity of the virus at high dilutions, since it has been shown that the infectivity of TMV is increased (about 66%) by filtration at ph 8.5 through bacteriological candles under negative pressure (Thornberry, 1935a). According to our data, filtration through bacteriological candles has no effect on the stability of TMV at high dilution. The data also confirm the previous report (Thornberry, 1935a) that filtration through a bacteriological candle increased the infectivity of the crude juice. The data do not suggest a mechanism of increased infectivity. Presumably, filtration either disaggregated clumps of virus particles or removed inhibitors. Inactivation of TMV by dilution might be suspected if the metals (Fe, Ca, Mg, and Cu) found in a highly purified preparation by Loring and Waritz (1957) are necessary for infectivity. An enzyme, L-leucine aminoexopeptidase, is inactivated by dilution in the absence of Mn++ or Mg++ ions, and the enzyme protein inactivated by dilution is reactivated by the addition of these ions (Smith, 1946; Roche, Thoai, and Bai, 1947). The data herein, however, clearly show that TMV is not inactivated by dilution and that the virus is actually infectious even at a 10-8 dilution when the solutions are assayed adequately. When the number of inoculated leaves (assay surface) was proportional to the dilution factor, the number of necrotic local lesions did not vary with the viral dilution. In view of these findings, the term inactivation with reference to viruses should be restricted to situations where the virus is known to be incapable of causing infection and should not be applied to situations where lack of infection is owing to limitations of the assay used for viral detection. The data, in addition to affirming the working hypothesis for this study, support the contention that a single viral particle is capable of infecting a susceptible site on a host. This support is not by direct evidence, but rather by refuting the claims that a "dose" of virus is necessary for infection. A "dose" of virus implies that a quantity of virus must be present at or make contact with the susceptible site. In this respect, concentration of virus in the inoculum is a critical factor, and a solution containing virus that is diluted beyond the end point of infectivity, as determined by the usual assays, would not be sufficiently concentrated to provide the required number of viral particles at a site to cause infection. Such solutions would, therefore, be noninfectious irrespective of the area of leaf surface (number of leaves) inoculated. In this study, however, the number of infection sites (local lesions) was irrespective of viral dilution when the assay surface (number of leaves) was increased in proportion to the dilution factor of the inoculum. Therefore, it seems that infection by TMV nucleoprotein is not strictly dependent upon concentration, but occurs as a chance phenomenon in which an individual rod infects the susceptible site. Since clumped particles would tend to disaggregate upon dilution, the virus would be dispersed as individual particles at the high dilutions. Dilutions in solutions of electrolyte would prevent an aggregation of a globulinlike protein, and filtration would remove viral aggregates. Thus, it seems evident that individual particles (presumably the 15 by 300 m,u rods) of TMV are capable of causing infection singly. ACKNOWLEDGMENTS The authors gladly acknowledged the technical assistance of Mary R. Phillippe and the critical review of the manuscript by W. M. Beaver. The junior author is grateful to the Graduate College for the graduate fellowship awarded to him. LITERATURE CITED AFANASIEV, M. M., AND H. E. MORRIS Bean virus 2 (yellow) on Great Northern bean in Montana. Phytopathology 42: BERAHA, L., M. VARZANDEH, AND H. H. THORN- BERRY Mechanism of the action of abrasives on infection by tobacco mosaic virus. Virology 1: KIM, W. S., AND D. J. HAGENDORN Streakinciting viruses of canning pea. Phytopathology 49: LORING, H. S., AND R. S. WARITZ Occurrence of iron, copper, calcium and magnesium in tobacco mosaic virus. Science 125: PATINO, G., AND W. J. ZAUMEYER A new strain of tobacco-streak virus from peas. Phytopathology 49: ROCHE, J., N. V. THOAI, AND N. K. BAI Sur la reactivation de la L-leucineaminoexopeptidase intestinale, par les ions manganese et

5 1326 THORNBERRY AND NAGAICH [VOL. 83 magnesium. Compt. rend. soc. biol. 141: SMITH, E. L Manganese and L-leucineaminoexopeptidase. J. Biol. Chem. 163: SMITH, K. M A textbook of plant virus diseases, 2nd ed. Little Brown & Co., Boston. STANLEY, W. M Isolation of a crystalline protein possessing the properties of tobaccomosaic virus. Science 81: STANLEY, W. M Chemical studies on the virus of tobacco mosaic. VI. The isolation from diseased Turkish tobacco plants of a crystalline protein possessing the properties of tobacco mosaic virus. Phytopathology 26: THOMAS, H. R Yellow dot, a virus disease of bean. Phytopathology 41: THORNBERRY, H. H. 1935a. Quantitative studies on the filtration of tobacco mosaic virus. Phytopathology 25: THORNBERRY, H. H. 1935b. Effect of phosphate buffers on infectivity of tobacco mosaic virus. Phytopathology 25: ZAUMEYER, W. J., AND G. PATINO Vein necrosis, another systemically infectious strain of alfalfa mosaic virus in bean. Phytopathology 50: ZAUMEYER, W. J., AND H. R. THOMAS Yellow stipple, a virus disease of bean. Phytopathology 40: Downloaded from on July 19, 2018 by guest