Inoculation and infection of leafhopper tissue cultures with a plant virus

VIROLOGY

30, (1906)

DISCUSSION

AND

PHELIMINARY

REPORTS

generate. Perhaps a more definite indication of virus infection is Mit’suhashi’s finding (2), in cultures inoculated with rice dwarf virus, No satisfactory technique has been de- of small “viruslike” particles 30 rnp in vised for inoculating plant tissue cultures diameter. These seem identical to those with plant viruses that is efficient enough found by Nasu (8) to be associatedonly with to permit studies of intracellular viral growth leafhoppers infected by rice dwarf virus. and other aspects of cell virus interaction However, the relation of such “virus-like” in the ways so widely employed in work particles to rice dwarf virus particles of 60with mammalian and avian viruses. With 80 rnp is not certain. Mitsuhashi’s finding (2) those plant viruses capable of multiplicaof rice dwarf virus particles of normal size tion in both plant and insect hosts, there is in the inoculated culture seemsinconclusive a possibility that the plant viruses may for he could observe “not many” and his have the same relationships to cultured illustrations show only particles 30 rnp in insect cells as do mammalian viruses to diameter. By contrast, in similar cultures cultured mammalian cells, with all the derived from the viruliferous insects, he obadvantages deriving therefrom. Recently served virus particles of normal size arranged there have been some reports that tissues in rows and enclosed,in sheathlike structures and organs from leafhoppers which transmit in “almost all cells.” plant viruses can be explanted to establish Working with wound-tumor virus (WTV), primary cultures. In two earlier reports we have successfully infected tissue cultures attempts have been made to inoculate such derived from the vector insect Agallia conprimary tissue cultures with plant viruses. styicta (Van Duzee) and have been able to Maramorosch et al. (1) reported that epi- detect the infection by staining with fluothelial cells of Macrosteles fuxifrons St%1 rescein-conjugated antibody, and by infecceased growing, developed granular inclutivity tests. These criteria of infection are sions, and finally deteriorated as a result of specific and unequivocal and our experiments inoculation with aster yellows virus. Howdemonstrate multiplication of the virus in ever, they mentioned no adequate comparithe inoculated tissues. No cytopathic effect son between the inoculated cultures and of the virus infection, including granulation, those subjected to comparable “inoculum” was observed under the light microscope. containing no virus. Their attempts to reInsect tissue cultures were obtained by cover infectivity in the inoculated cultures explanting embryo tissue fragments disby injecting vector insects were not successsected from eggs of virus-free A. constricta ful. 1Jitsuhashi (2), working with rice dwarf laid in crimson clover petioles 6 or 7 days virus and primary cultures of its leafhopper earlier. This material was selected because vector, Nephotettix cincticeps Uhler, observed Hirumi and Maramorosch (4) demonstrated changes in cell appearance in the inoculated that embryos of this age are a better source cultures similar to those observed by Maraof tissue cultures than older and younger morosch et ab. (1). However, Mitsuhashi con- embryos. The eggs were first removed from sidered that these changes are not specific the petioles and surface disinfected by dipand are often seen when leafhopper tissue ping in 75% ethanol for 1 minute and then cultures from virus-free insects begin to de- in 0.1% HgClz for 3 minutes, followed by a seriesof 5 washings in sterile water. Young 1 This research was supported in part by grants embryos were dissected out on depression from the National Science Foundation (GB-2020) and the National Institutes of Health (AI-06392). slides in modified Tyrode’s solution containinoculation

and Infection Cultures with

of leafhopper a Plant Virus’

Tissue

562

DISCUSSION

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PRELIMINARY

ing 5 mg/ml bovine serum albumin-the addition of bovine serum albumin helped overcome the floating of embryos often encountered at this step. The dissected embryos, freed from yolk and cut into a few pieces, were trypsinized for 15-20 minutes at room temperature in 0.25 % trypsin in Rinaldini’s citrate-salt solution (5). They were then transferred in groups of several piecesto a drop of growth medium on 25-mm coverslips fittable to Sykes-Moore culture chambers. The brief trypsinization period did not result in much cell dissociation but helped improve the initial outgrowth of cells from the explants. The basic culture medium of Mitsuhashi and Maramorosch (6) supplemented with 20% fetal bovine serum and antibiotics gave more satisfactory results than several other media tried and was used for both cell growth and virus propagation. Cultures 10 days old or more were selected for inoculation. These contained the original embryo tissue explants surrounded by well developed cell sheets in which an epithelial cell type was dominant. Other types of cells as described by Mitsuhashi and Maramorosch (6) could also be found in the mixed cell population. Inoculation was carried out by replacing growth medium with inoculum and allowing an adsorption period of 2 or 3 hours at room temperature. Inocula were prepared from adult leafhoppers, except where otherwise stated. These had acquired virus by feeding for 2 or 3 days as young nymphs on crimson clover, Trifolium incarnatum L., infected with WTV. They were then maintained at 27 f 2” for 4 weeks on healthy crimson clover in cages in a growth chamber provided with 14 hours of light at 800 footcandles each day. Reddy and Black (7) had shown that infective virus reaches a peak in such insects 28 days after the start of the acquisition period. Usually 3&60 viruliferous insects were homogenized in 2 ml of the growth medium with a reduced fetal bovine serum content (5 %). The homogenates were used after clarification at 3100 Qfor 5 minutes and after successivepassages through Millipore filters having pore diameters of 0.65 and 0.30 ~1. For controls, comparable cultures were

REPORTS

563

exposed during the adsorption period to homogenates similarly prepared from virusfree insects. Both inoculated and control cultures were subsequently incubated at room temperature (24”); the medium was replaced every 2 or 3 days. The protective solution (0.1 M glycine and 0.01 M MgCl,) routinely employed in this laboratory to suspend WTV had to be avoided as a virus diluent in infecting tissue cultures because the chelating property possessedby the glycine tends to cause cell detachment from the glasssurface. Fluorescent Antibody Staininy. The inoculated tissue cultures were examined for evidence of WTV infection by the direct fluorescent antibody technique of Coons as adapted to the study of WTV by Nagaraj et al. (8) and Sinha and Reddy (9). In brief, the procedure consisted of drying coverslip cultures either unwashed or washed with phosphate-buffered saline (0.01 M potassium phosphate, 0.15 IM NaCl, pH 7.0), for 10 minutes at 35”. The cultures were then fixed in ether or acetone for 5-10 minutes, stained for 25-30 minutes in WTV-antiserum conjugated with fluorescein-isothiocyanate, thoroughly washed in buffered saline and mounted in the same saline containing 10% glycerol. Control cultures which had been exposed to the healthy insect homogenates were always included for comparison with inoculated cultures. In experiments in which one or more coverslip cultures were stained on various days with the conjugated WTV antiserum, the first evidence of viral antigens was consistently noted in a few cells in inoculated cultures on the third day after inoculation. The intensity of staining and the number of stained cells increased on later days. The viral antigens appeared as discrete brilliant yellow greenish spots in the cytoplasm. Such staining was not obtained in control cultures. The specific yellow greenish spots were not observed in nuclei in any infected cultures examined. The cytoplasmic location of WTV antigens as revealed in inoculated tissue cultures in this study is in agreement with previous observations by Sinha (10) and Shikata and Maramorosch (11, 12). These investigators, however, employed cells from insects that were viruliferous when they were dissected.

564

DISCUSSIOK

AND

PRELIMINARY TABLE

RESULTS

OF INFECTIVITY

Sample

REPORTS

I

TESTS ON ‘.LIEDIUM .~R‘D TRITCR.~TED CELLS T.4KEN FROM TISSPE CULTURES INO~UIA~ED IVITH WTV

Number of insects surviving injection after 21 days

Inoculum Cell samples* O-time, inoculated 4-day, inoculated B-day, inoculated B-day, uninoculated control Medium samples from inoculated cultures O-time 2-day 4-day B-day B-day

Soluble antigen titer in insects injected with samples

30

841

Assay of relative virus concentration in sample

AT Va~tror;s

TIMES

Calculated” relative residual virus concentration from original inoculum

107.5

37 45 46 28

178 401 611 Negative

105.2 106.4 107.o Negative

43 43 55 55 38

NegativeC 317 205 388 388

Negative 106.0 105.4 106.3 108.3

-

105.’ 105.7 105.’ 104.5 103.9

a Medium samples were taken from two inoculated cultures and pooled before injection. Both the zero-time and 2-day medium samples came from the same medium, which replaced inoculum after the latter had been removed and the inoculated cultures washed twice with PBS-Mg. The zero-time sample was taken 3 hours after completion of inoculation, and the other 2 days later. All later medium samples were taken after one or more medium changes. It is conservatively estimated that each washing with PBS-Mg or each change of medium must have removed N of the virus so that the residual virus concentration after each such operation was x (or 10-O.“) of what it was before. The concentration of residual virus from the inoculum would have been reduced to the different levels indicated for the different medium samples in column 5. b In each case tissue explants and cell sheets from four cultures were first washed and then triturated in 0.16 ml id PBS-Mg before assay. c The limit of sensit,ivity of the infectivity test is in the neighborhood of a relative virus concentration of 104.

Infectivity Tests. Further evidence of WTV infection was provided by infectivity tests made on the inoculated cells or the medium in which they were grown. The relative concentration of infective WTV (7) was calculated from the results of precipitin ring-time tests (IS, 14) on extracts from injected insects. To detect virus infectivity associatedwith cells alone, tissue explants and cell sheets were washed twice in Dulbecco’s phosphatebuffered saline modified to contain 0.005 1M MgClz (PBS-Mg). Then they were frozen for 30 minutes at -25” and thawed to 2”. The ruptured cells, from a pool of four cultures, were ground in about 0.16 ml PBS-Mg on a depression slide. Such preparations made from cells immediately after inoculation and at later times were injected into large healthy nymphs. In addition, infective

virus released from living cells into the medium was measured by taking samples of medium at intervals and testing them similarly by injection into healthy nymphs. Usually 45-60 insects were injected with each sample. The relative infectivity, for convenience referred to below as relative virus concentration, was determined by the method described by Reddy and Black (7) and is based on their dilution curve and scale of relative virus concentrations. The absolute numbers of virus particles at the different concentrations is not known. Table 1 presents the measurements of WTV infectivities associated with cells and medium in one experiment. Although the relative virus concentration detected in the cells under the experimental conditions did not exceed the level in the original inoculum, there was an obvious increase in infectivity

DISCUSSION

AND

PRELIMINARY TABLE

SERIAL

Passage no.

Source

of inoculum

Sweet clover tissues Passage 1 Passage 2 Passage 3 Passage 3 Passage 5 Passage 6 a NT,

not

tumor

PASSAGE

Days between passages

8 9 11 9 11 8

OF WTV

565

REPORTS

2

IN VECTOR

TISSUE

Number of Soluble insects antigen titer surviving in insects injection injected with after 21 days inoculum 43

430

NTa 42 40 NT 46 32

NT 277 317 NT 208 242

CULTURES

Assay of relative virus concentration in inocuium

n^l,...l..r^J -.,,.r:..* LdAALlaLc” IC:ldl”C residual virus concentration from original inoculum

106.5

105.7 106.’ 105.4 105.6

105.o 103.5 102.0 108.5 10-1.0 10-2.5

tested.

as the inoculated cultures were incubated. The virus concentration, detectable in the medium, seemedto reach a plateau level in about 2 days after which no significant increase in infectivity was obtained. This is not unexpected, for WTV has been found to be unstable at room temperature. In an experiment with a sample of WTV dispersed in the medium, the relative virus concentration after 0, 2, 8, and 24 hours at 24” was 10s9 106.3. 105.“, and 104.6, respectively. In other words, after 24 hours the concentration of infective virus had been reduced to almost the minimal level detectable by the method employed, namely about 104.At the plateau level the rate of virus release from cells and the rate of virus inactivation are probably approximately equal. Virus increase detectable in the medium is obvious if we take into consideration dilutions introduced by washing the cultures and changing the medium. As described in footnote a, Table 1, the routine of changing the medium with washings reduced the virus concentration by at least 1.8 log units at the end of the inoculation period. The relative virus concentrations of inoculum and corresponding zero-time medium (Table 1) indicate that the actual reduction was 103.5, the difference between 107.5 and the negative result interpreted as 104. In two other tests the corresponding drop in concentration was from lO*.l to 104.0and 1O7.4 to another negative interpreted as 104. The estimate of 0.6 log unit for each washing or

medium change, used in calculating the hypothetical values in column 5, is therefore very conservative. After the infectivity plateau is reached, the differences between the values in column 4 and values for corresponding times in column 5 clearly indicate virus multiplication. Serial passage of WTV in cultures. A serial passage experiment was designed to obtain further evidence of WTV multiplication in tissue cultures. Root tumors on sweet clover, Melilotus oficinalis (L.) Lam., clone GO, infected by WTV isolate VIee were used as a source of inoculum for infecting healthy tissue cultures. The inoculum was prepared by grinding 0.5 g of tumor tissue in 1.5 ml of 0.05 M KtHP04 containing 0.01 M Na303. To the pulp was added 8 ml of protective solution (0.1 M glycine and 0.01 M MgCl,). The virus sample, clarified and filtered successively through Millipore filters of 0.65 p and 0.30 CLdiameter, was further diluted to 1: 10 with medium before being used to inoculate tissue cultures. Serial passageswere carried out at S- to 11-day intervals. Usually, four cultures were inoculated in each passage and two were retained uninoculated as controls. In each passage, tests for virus were carried out on one control culture by staining with fluorescein-conjugated antiserum and on the other by injecting an undiluted triturate of the culture into leafhopper nymphs. All these tests were negative. Of the four inoculated cultures in each passage, one stained

566

DISCUSSION

ANI)

PRELIMINARY

REPORTS

with conjugated antiserum revealed t,he tissue cultures with plant, virus samples predevelopment of specific antigens. The other pared directly from infectsed inSects, infected three, with medium unremoved, were frozen, insect, tissue ctultures, and infected plant thawed, triturated, and clarified. The three materials by dilution in appropriate solvents. cultures yielded 0.12 ml of a solut,ion which was mixed with 3.72 ml of fresh medium to give a dilution of 1:32 for inoculation of We wish to express our appreciation to Dr. the next passage. Such inoculum was also George K. Hirst for the opportunities he extended used to inject healthy nymphs in infectivity to the senior author to study animal tissue culture tests. Since the original inoculum had a techniques in his laboratory before this research and particularly for the tutelage relative virus concentration of 106.5 and was undertaken by 1)rs. Robert W. Simpson and Marcel W. Pons. since each transfer resulted in a 1:32 (or 10-1,5) dilution of virus in the original REFERENCES inoculum, the series of passages should have reduced the concentration of virus from 1. MARAMOROSCH, K., MITSGHASHI, J., STREISthe original inoculum to 1O-2.5 at the end of SLE, G., and HIRKJMI, H., Bacterial. Proc. 1965 (Abst. of 65th Ann. Meeting Am. Sot. the seventh passage. This concentration is Microbial.), p. 120. well below that detectable by the infec2. MITSUHASHI, J. Japan., J. Appl. Entomol. tivity test’. However, the experimental Zool. 9, 137-141 (1965). results (Table 2) show no such gradual 5. NASU, S., Proc. Conf. Relationships between virus reduction in the cell samples. Instead, Arthropods and Plant-Pathogenic Virus, at each passage in which an infectivity test Japan City Center, Tokyo, Oct. 2528, 1965, was attempted, virus was recovered at United States-Japan, Scientific Cooperation about the same concentration from the cell Program, Publication 1: 91-96. samples, that is, 105.4 to 106.1. The data 6. HIRUMI, H., and MARAMOROSCH, K., Science prove multiplication of virus during the 144, 1465-1467 (1964). series of passages. 5. RINALDINI, L. M., Exptl. Cell Res. 16, 477-505 (1959). WTV appeared to produce no cytopathic 6. MITSUHASHI, J., and MARAMOKOSCH, K ., effects on the cultured cells of its vector Contrib. Boyce Thompson Insf. 22, 435-460 leafhopper, A. constricta. In comparison with (1964). control cultures no apparent changes in the 7. REDDY, I>. V. R., and BLACK, L. M., Virology inoculated tissue cultures could be detected 30, 551661 1966. under the phase contrast microscope. One A. N., SINHA, R. C., and BLACK, 8. NAGARAJ, culture continued to proliferate until 23 L. M., Virology 15, 205-208 (1961). days after inoculation when it was stained R. C., and REDDY, D. V. R., Virology 9. SIXHA, with the fluorescein-conjugated antiserum 24, 626-634 (1964). to WTV and revealed an intense specific 10. SINHA, R. C., Virology 26, 673-686 (1965). E., and MARAMOROSCH, K., Nature staining. A second culture was used 4 days 11. SHIKATA, 208, 507-508 (1965). and a third, 8 days, after inoculation to E., and MARAMOROSCH, K., Virology establish new cultures by transplanting the 12. SHIKATA, 2i, 461-475 (1965). tissue explants, and the remaining cell sheets 1s. WHITCOMB, R. F., and BLACK, L. M., virology were stained and found to contain WTV 13, 507-508 (1961). antigens. The transferred explants conR. F., ViroZogy 24, 488492 (1964). 14. WHITCO~~B, tinued to grow and yielded cell sheets until REN-JONG CHIU 45 days and 64 days after inoculation when I). V. R. REDDY the tissue explants in the cultures were again L. M. BLACK removed. The cell sheets again took the Department of Botany specific staining with conjugated antiserum. University of Illinois Incidentally, these experiments demonUrbana, Illinois strated successful inoculation of leafhopper Accepted July 27, 1966