Absorption and translocation of mammalian viruses by plants

VIROLOGY 6, 623-636 (1958)

Absorption

and

II. Recovery WILLIAM Deparhen.t

Translocation of Mammalian by Plants and Distribution

H. MURPHY,

of Uacferiology

of Viruses in Plants1p9

,JR.~ 4~11 JEROME

and Zrwr~nnology, dccepted

July

Viruses

University

T. SYVERTON of Minnesotn,

Minneapolis

1, 1958

Pea, tomato, potato, and lett’uce plants in modified hydroponic culture were exposed to strain FA mouse encephalomyelitis virus or to type 1 (Mahoney) or 2 (YSK or MEF-1) polioviruses, under conditions permitting independent tests for absorption and translocation. FA virus regularly entered plant roots and attained significant concentration; acropetal translocation occurred irlfrequently. Type 1 poliovirus as mixed suspension of monkey brain tissue extract and cell culture virus was absorbed by tomato plant roots but, not translocated to aerial parts; Mahoney and YSK polioviruses in tissue culture fluid, and MEF-1 poliovirus in mouse brain suspension, were neither absorbed nor translocated by plants. In summary of positive experiments, virus was recovered from 47 of 52 plant roots and from t,he aerial portions of 6 of the 52 plants. Evidence is presented that instances of virus absorption and translocation by plant,s did not in fact originate from (a) accident,al contamination, (b) act,ivation of latent virus in test mice, or (c) capillary ascension of virus along root exteriors. INTRODUCTION

Evidence given in a preceding report (Murphy ef al., 1958) demonstrated that mouse encephalomyelitis virus and poliovirus survived under environmenial conditions requisite for possible absorpt,ion by plant roots. This report describes experiments t,hat concern absorption and translocation of mouse encephalomyelitis virus and poliovirus by plants. 1 Aided by a Grant from The National Foundation for Infantile Paralysis, Inc. * The material in this paper represents in part t,he thesis submitted by William H. Murphy, Jr., in partial fulfillment of the requirements for the Ph.D. degree. 3 Present address: Department of Bacteriology, University of Michigan School of Medicine, Ann Arbor, Michigan. 623

624

MURPHY,

MATERIALS

JR.,

AND

AND

SYVERTON

METHODS

virus

Pools of strain FA mouse encephalomyelitis virus and polioviruses type 1 (Mahoney) and type 2 (YSK and MEF-l), were prepared as indicated previously (Murphy et al., 1958). Plants Plants were grown in a greenhouse or in a 75’ F incubator with controlled 15-hour daily photoperiod provided by fluorescent and clear incandescent lamps. Plant growth procedure was described previously; sufficient oxygen for root growth was provided by daily renewal of nutrient solution. The plants employed for these studies were the “Dwarf Champion” variety of tomato, “Early Alaska” variety of pea, and “Great Lakes” variety of head lettuce. Virus Assays of Plant Extracts Portions of each plant were tested for virus: (a) roots in the pot, (b) lower two-thirds of stem and leaves, and (c) upper third of the plant, principally leaves. Each portion of a plant was taken with a separate set of sterile instruments, frozen at -2O’C for at least 24 hours, thawed rapidly (Bawden and Pirie, 1951), and triturated with sterile alundum. During trituration, a volume of sterile diluent (D) equivalent to plant tissue weight was added. Diluent consisted of normal monkey serum inactivated 30 minutes at 56’, 10% in Hanks’ balanced salt solution (H100). Supernates of triturates spun at low speed were recentrifuged at 13,000 g for 30 minutes to sediment bacteria and fungi. Final supernates diluted 1: 10 with D were stored in sealed ampoules at -20’. Virusassay procedures were carried out as before (Murphy et al., 1958). Cellculture methods for isolation and identification of polioviruses have been recorded by Syverton et al. (1954). RESULTS

Tests for Toxicity of Plant Extracts for FA Virus, Polioviruses, and HeLa Cells Extracts of normal tomato, potato, and pea tissues (roots, stems, and leaves) were tested for virucidal activity (Chantrill et al., 1952) and influence on detection of types 1 and 2 polioviruses. Decimal dilutions of viruses in H-100 were mixed in equal volume with plant extracts. Extracts of pea plants, by reason of greater mouse toxicity, were diluted

ABSORPTION

AND

TRANSLOCATION

TABLE

OF MAMMALIAN

VIRUSES.

II

625

1

EFFECT OF PLANT EXTRACTS ON MOUSE ENCEPHALOMYELITIS (FA) AND TYPE 2 POLIOYYELITIS (MEF-1) VIRUSES Virustiter Virus

H-100b control

after exposure’l to extract of:

Tomato stems and leaves

Potato stems and leaves

Pea stems and leaves

II Virus was incubnted with extract in equal volume for 2 hours at room temperat,ure. b H-100: balanced salt solution incubst.ed in equal volume with virus.

1:5 with D before addition to viruses. These mixtures, together with control virus suspensions brought to equivalent concentration with H100, were incubated at room temperature for 2 hours. Samples of 0.03 ml each of FA and MEF viruses were injected intracerebrally into 21day Swiss albino mice. Each of ten HeLa tube cultures containing 0.8 ml of maintenance medium was inoculated with O.l-ml aliquots respectively of D, or plant extract, and/or decimal dilutions of type 1 or YSK poliovirus. Plant extracts were not virucidal for FA or MEF-1 viruses (Table 1) under condit)ions of experiment. The apparent enhancement of FA virus infectivity by pea extract was not explored further. Separate extracts of roots, stems, and leaves of tomato, potato, pea, and lettuce plants, each inoculated into HeLa cell cultures without or with from 10 to 500 TCID60 of virus, did not delay appearance of cytopathogenicity induced by type 1 or YSK polioviruses, reduce sensitivity of cell cultures to low virus doses, or affect’ cellular morphology in comparison with cont’rolx. It was concluded from these observat,ions that tests for polioviruses in plant materials would not, be hindered by toxicity of extracts for viruses, mice, or cells. Tests for Absorption and Tramlocation

of FA Virus

Three-week-old pea plants (with roots protruding from bottoms of pots) were divided into four groups. Two groups, one with intact roots and the other wit,h roots severed transversely, were exposed to FA virus (1% mouse-brain suspension) diluted in nutrient solution to final titer of 10-4.7. Fresh virus in plant nutrient solution was sdded at 4-day intervals to the roots of each plant. A third group with intact roots was exposed to virus (final titer lo-*) purified as described by Weil et at. (1952) ;

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2

ABSORPTION AND TRANSLOCATION OF STRAIN FA MOUSE ENCEPHALOMYELITIS VIRUS BY PLANTS

Experimental

conditions

Pea plants: roots

normal

Pea plants: roots

Pea plants: roots

Exposure of plants lo virus in days

Virus

2 4

Mouse Mouse

brain brain

0/4c O/4

3/4

l/4 O/4

O/l O/l

severed

2 4 6 8

Mouse brain Mouse brain Mouse brain Mouse brain

O/4 O/4 014 O/4

4/4 J/4 4/4 4/4

114 O/J l/4 l/4

O/l O/l O/l O/l

normal

4 7

I’urifiedd Purified

‘J/6 O/6

‘J/f3 O/f3

O/6 O/6

o/2

4

Mouse

brain

O/J

114

l/4

O/l

4

Mouse

brain

O/4

3/4

O/4

O/l

1 4

Mouse Mouse

brain brain

O/4 O/l6

2/4 lo/16

O/4 2/16

O/l O/4

Potato plants: ma1 roots Tomato plants: ered roots Tomat,o plants: ma1 roots

nor-

sev-

nor-

w

o/2

a Soil surrounding roots in pots was test,ed to det,ermine whether virus was restricted to internal root tissues. b Plants exposed to uninfected mouse-brain suspension in nutrient solution. c Numerator indicates number of extracts yielding virus; the denominator, number of extracts tested for virus by intracerebral injection of Swiss albino mice. d Virus was “purified” by t,he procedure described by Weil et al. (1952).

nutrient solution containing virus for these plants was changed on alternate days. The fourth group of control plants was exposed to nutrient solution containing equivalent concentration of normal mouse brain suspension in place of infected brain suspension. As before, pea plant extracts were diluted before inoculation of mice. An experiment with 3month-old tomato and potato plants was performed similarly except that purified virus was not tested. Virus assays of extracts of surrounding soil, roots, and pooled stems and leaves, revealed that purified virus was not adsorbed by pea plant roots (Table 2); severing of roots facili-

ABSORPTION

AND

TRANSLOCATION

OF MBMMz4LIAN

TABLE CONCESTRATION TYPE

OF STRAIN 1 POLIOVIRUS

VIRUSES.

II

G27

3

FA Mouse:

ENCEPHALOMYELITIS IN EXTRACTS OF REPRESENTATIVE PLANT TISSUES

VIRUS

AND

Exposure Origin

Norm& pea roots Severed pea roots Leaves from peas with normal roots J,eaves from peas with severed roots Normal tomato roots Leaves from tomatoes wit,h norma1 roots Normal t,omato roots Normal tomato roots Normal tomato roots Normal tomato roots

Negative log of pl+s titer of virus to vLrus in plant extract III days

Virus

of tissue extract

Type Type Type Type

1 1 1 1

FB FA FA

2 2 2

1.2h 2.7 0.8

FA

4

1.1

FA FA

4 4

2.5 1.0

poliovirusc poliovirus poliovirus poliovirus

2 4 6 0

1.5 2.2 3.0 2.5

n Plant roots were not severed for exposure to virus. 6 Titer of virus as derived from tissue extracts of six plants; there was no experimental basis to make assumptions concerning extent of dilution or inactivntion of virus in plant tissues. c l’oliovirus preparations used for exposure’to plants consisted of infected brain tissue reinforced wit,h HeLa cell culture virils.

tnt,ed association with virus. Virus was recovered from aerial port,ions of pea, tomato, and potato plants, but less frequently than from rook. Failure to recover virus from control plants showed that positive virus assays did not result from activation of latent virus in test mice. Representative plant extracts were assayed for virus content by injection of decimal dilut,ions into mice; Gters were calculated as in previous st,udies (Murphy et nl., 1958). Virus concentration was higher in roots than in leaves (Table 3). Tit,ers were considered suficiently high to exclude accidental cont,amill:lt,ioll as a likely source of isolated virus. Prevention

!I!J A%hwne OS(‘apillar!/

A~scmsion of

FA TTirrlson Plant Roots

To test the possibility of virus migrat)ion by capillary ascension along root ext,eriors, a preventive procedure was devised. Ten tomato plants were prepared as usual in pots containing soil so that roots probruded through the opening in the pot bottom, except that Dow-Corning silicone

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FIG. 1. To prevent the capillary ascension of virus preparations along root exteriors, silicone stopcock grease was packed into the orifice of pots as indicated. to determine its distribution in plant tisEosin Yvas added to a virus preparation sues. A fter exposure to the dye-virus preparation for 4 days the plant was disinteriors of roots and the lower stems and sected; dye was found only within leaves.

stopco ck grease was packed into the opening to surround the roots (Fig. 1). Plztnts in pots so prepared were set in beakers containing nutrient solutic In with 1% normal mouse brain suspension added. Eosin was added to the nutrient solution to final 0.1% concentration. This dye was used on the ) assumption that cellulose content of roots in hydroponic culture

ABSORPTION

AND

TRANSLOCATIOS

TABLE ABSORI'TION

Tomatoes Potatoes

AND

OF MAMMALIAS

629

II

4

TRAMLOCATION OF STRAIS FA $IOUSE VIRCS BY SILICONE-TREATED PLANTS

1 4 4

VIRUSES.

o/4*

2/4b

O/8

4/8

O/J

l/J

O/4 O/S O/4

I~NCE~~HALO~\IYELIT~S

O/J

l/8 l/4

O/l o/2 O/l

‘I Soil surrormding roots in pots was test,cd to determine whether virlls was I’Pstricted t,o internal root tissues. * Numerat,or indicates number of ext rarts yielding virus; denomin:tt,or, nrlmhrr of extra& tested for virlls by int,racerel,r:tl injection of Swiss :ttt)ino mice.

would produce a negative charge on root exteriors: a negat,ively charged dye should ascend by capillarity most readily along root exteriors so charged. After exposure of planbs to dye-brain suspension for 4 days, dye was found only in the interiors of potted portions of roots. It, was concluded that silicone could be used to exclude capillary ascension of virus. Three-month-old tomato and 2-month-old potato plants were prepared in potted soil with roots protruding through silicone-packed orifices. Exposed roots were immersed for 4 days in beakers containing nutrient solution supplemented with FA virus as 1% brain suspension, as before. Virus was recovered from roots and one extract of leaves from each plant type, in spite of silicone-blocking of root, exteriors (Table 4). It is noteworthy t,hat virus could be absorbed by roots within 24 hours after exposure, since t,his interval seems too short for significant, nheration of root integrity by microbiologic activity. MentQicafion

of Viruses Recowred ,from Plant Tissue

Preliminary experiments confirmed reports (Dean, 1951; Daniels et al., 1952) of difficulty encountered in tests for neutralization of mouse encephalomyelitis virus. Such difficulty was circumvented by passive immunization of mice used for assay of neutralization. Potent antiserum t,o FA virus was obtained from two monkeys bled by cardiac punct,ure 1.5 days after final injection of four intramuscular doses of 1.0 ml of antigenic mat,erial, given at l&day intervals t,o each monkey; for injection, 10% infected mouse brain suspension was emulsified in ndjuvunt (Salk et al., 1951).

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MURPHY,

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TABLE

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5

?~JEUTRALIZATION OF REPRESENTATIVE \r~~~~ STRAINS RECOVERED FRO&X ExTR.~CTS OF Pr,a~~s EXPOSED TO MOUSE ENCEPHALOR~~ELITIH VIRUS, STRAIN FA, BY MONKEY ANTISERUM TO PROTOTYPE VIRUS Tissue-extract

virus source

Neutralization

Pea Z-leaves Potato 2-roots Potato 2-leaves Tomato 14.leaves Tomato Wroots Control

virus-vs-homotypic

index

113 113 127 76 100 serum

100

Decimal dilutions of FA virus as 20% brain suspension, or of virus derived from plant extracts, were incubated for 2 hours at 37” and 18 hours at 4’ with equal volumes of undiluted heat-inactivated normal monkey serum or antiserum to FA virus, then were injected in 0.03-ml amounts intracerebrally into 21-day Swiss albino mice, six per dilution. On second and tenth days following, test animals received 0.1 ml of FA virus antiserum intraperitoneally; control animals similarly received normal monkey serum. Neutralization indexes were calculated after 15 days of observation. Significant neutralization indexes (greater than 50) showed that viruses recovered from plants were derived from FA virus to which plants were exposed (Table 5).

Translocation of FA Virus in Tom,atoPlant Stems Results of preceding experiments, in which virus was recovered less frequently and in lower concentration from stems and leaves than from roots, suggested that virus was transferred acropetally from plant roots only with difficulty. To test for acropetal transfer, apical portions of twenty 3-month-old tomato plants were cut transversely, placed in nutrient solution in 15 X 150-mm tubes, and incubated until adventitious root formation began in about 3 days. Nutrient solution for sixteen plants was supplemented with 1% suspension of FA virus and changed every fourth day; four virus-exposed plants and one unexposed plant were taken at intervals of 2, 3, 5, and 10 days, and extracts made of upper stems and leaves. Assays of extracts for virus indicated that only one (representative of 5-day exposure) of all extracts contained virus (Table 6). These findings supported the conclusion that virus could be absorbed by roots without being readily translocated to stems and leaves.

ABSORPTION

BND

TRANSLOCATION

TABLE RESULTS

OF MAMMALIBN

II

631

6

OF STUDIES ON THE TRANSLOCATION IN SEVERED TOMATO STEMS

Exposure of serered stems to virus in days .-

VIRUSES.

OF FA VIRUS

Recovery of virus from: Pouled stems and leaves

Control plants”

a Severed stems exposed to uninfected mouse brain t.issue suspensions in nutrient solution. h Numerator indicates the numhrr of extracts yielding virus; t.he denominrttor, the number of extracts tested for vinls hy intracerebral inoculation of Swiss albino mice.

Absorption

and Translocation of Tissue Culture Poliocirus

Roots of plants were immersed in beakers containing nutrient solut,ion supplemented with 1% of fluid containing type 1 poliovirus (final titer 10-5.6), or with YSIi poliovirus fluid (final titer of 1O-4.8)of tissue culture origin. Viruses had been propagated in HeLa cell cult,ures. Eight lmonth-old tomato plants, and eight 2-mont’h-old potato plants, supported in pots wi-ith orifices charged with silicone, were exposed for 12 days t,o virus suspensions changed every third day. A group of eight 3-month-old tomato plants not treated with silicone also was exposed to virus. During experiment,s, water was added to beakers to replenish fluid lost by transpiration (50-90 ml per day). Assny of nutrient solution surrounding roots showed that infectious virus was present throughout the test period. Virus was not recovered from extracts of soil, roots, stems, or leaves. Assays of extracts from control plants not exposed to virus were negative as usual. l’olioviruses grown in Cssue culture thus neither were absorbed by root,s nor ascended roots by capillarity. A bsorption and Translocation of I’olioCxs

in Brrrin Slrspcnsion

To t,est, the role of poliovirus menstruum on absorption and translocation, a 1% suspension of infected monkey brain reinforced with type 1 poliovirus derived from HeLa cell culture was employed. Roots of tomato plants, int,act or severed transversely, were exposed to nutrient solution supplemented with virus brain-tissue suspension. Intact roots of %-month-old tomato plants were exposed t-o solut,ion containing type 1 virus to final titer of 10-4.7 (for group A, tested in 1955) or lo-“.” (for

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TABLE

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7

ABSORPTION BY TOMATO PLANTS OF TYPE 1 I’OLIOVIRUS Experimental

conditions

IN BRAIN

SUSPENSION*

Exposure of plants to virus in days

Roots

Recovery of virus from: Stems

Leaves

Control plants

2 3 4

l/4 2/4 3/4

o/4 o/4 o/4

O/4 o/4 o/4

O/l O/l O/l

l/5 4/5 415 5/5 4/4

o/5 o/5 o/5 o/5 o/4

o/5 o/5 o/5 o/5 o/4

O/l O/l O/l O/l O/l

Tomatoes; group Ab Severed roots Normal roots Normal roots Normal roots Tomatoes, Normal Normal Normal Normal Normal

group I3 roots roots roots roots roots

u One per cent infected monkey brain tissue reinforced with poliovirus derived from HeLa cell cultures. b Plants of group A were exposed and tested for virus during summer of 1955; those of group B were tested during the summer of 1956. c Numerator indicates number of extracts yielding virus; denominator, the number of extracts tested.

group B tested in 1956 under similar conditions). Fresh virus suspension was added every third day. Results for tomato plants (Table 7) showed that type 1 virus was taken up by roots in 48 hours, but was not translocated to stems or leaves even after 9 days of incubation. Intact roots did not accumulate significantly increased concentrations of virus after 3 days. Extracts of stems and leaves of tomato plants and extracts of control plants made in from 2 to 9 days of exposure were uniformly negative on assay for virus, although infectious virus persisted in solutions surrounding roots throughout the test period. Association of brain-tissue elements with virus appeared to facilitate absorption by roots but not translocation. Three-month-old tomato plants were exposed to mouse brain suspensions of MEF-1 virus (final titer 10-1.7) under conditions described above. Soil, plant extracts, and nutrient solutions were assayed with mice as described for FA virus (MEF-1 virus employed was not cytopathogenic for HeLa cells) ; results were uniformly negative. The experiment was otherwise informative because test mice did not yield mouse encephalo-

ABSORPTION

AND

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OF MAMMALIAS

VIRUSES.

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633

myelitis virus under the same conditions of virus assay as used for $‘A virus. These findings support the conclusion that FA virus apparently absorbed and translocated by plants was, as indicated by negative coutrols, not derived from test mice. It appeared that translocation observed for FA virus, but not seen with poliovirus, was not dependent on virus association wit,h brain-tissue elements. 4 bsorption and Translocation of Poliovirus from Soil A study was made of the capacity of type 1 poliovirus to gain entry from soil into the roots of lettuce, tomato, and potato plants. Plants were seeded in pots containing 4 parts sand and 1 part soil. Four-month-old t,omato, pot,ato, and lettuce plants \yere prepared for test by insertion of a 10 X 200-mm glass tSube into surrounding soil for addition of fluid. Soil was covered with parafilm to prevent, contamination of lower plant leaves with virus. Water or nutrient solution was added to soil as needed. Solution supplemented to 2% concentration with tissue cukure fluid containing type 1 poliovirus (final titer 10-4.7) was added to soil surrounding lettuce plants in 20-ml amounts every third day; solution containing infected tissue culture fluid to 1% concentration was added at S-day intervals in 50-ml amounts to tomato-plant soil. Three-month-old potato plants also were tested: plants of one group received daily for 10 days 50 ml of solution cont,aining 2% of infected culture fluid, those of another group received every third day for 15 days 100 ml of solution containing 1% of infected culture fluid. Four test, plants and one control plant were harvested at intervals up to 10 days for potat,o plants in group A, 15 days for potato plants in group B, and at intervals of 4 days to 1G days for lettuce and t’omato plants, and each lot was extract’ed. Separate cellculture assays of stems and leaves of test plants and of control plant8s for virus were uniformly negative. Assays of soil showed that virus was present around each t,est plant during the period of experiment. Type 1 poliovirus was not absorbed and t,r:mslocated from soil by the plant,s under these conditions. DISCUSSIoN

These studies with representative encephalomyelitic viruses indicated that absorption by pea, tomato, or potato plant roots could occur fairly readily under experimental conditions; acropetal translocation was observed only with mouse encephalomyelitis virus, and infrequently. Xegative findings for MEF poliovirus, and rare induction of spontaneous infection in assay mice by injection of plant extracts, established that

634

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SYVERTON

experimental procedure was adequate to insure that FA virus recovered from roots or upper parts of plants had indeed been absorbed and translocated. Evidence likewise excluded accidental contamination as a source of recovered virus, and dye test indicated that application of silicone about roots was sufficient to block capillary ascension of virus along root exteriors. Absorption of ovalbumin by plant roots and acropetal translocation to stems and leaves (Chester, 1937), absorption and translocation by plants of phytopathogenic viruses, and results of preliminary experiments in this Department (Skarnes, 1952), suggested that’ systematic investigabion of absorption and translocation by plants of encephalomyelitic viruses might be academically and epidemiologically interesting. Results of study here reported suggest that such movement of virus in nature is not impossible, but probably does not occur with sufficient regularity to be important as a mechanism for transmission or for interepidemic survival of poliovirus. The observed irregularity in translocation of virus from roots to aerial parts of plants is consistent with similar findings for phytopathogenic viruses (Ii’ajardo, 1930; Bennett, 1940; Fulton, 1941; Capoor, 1949; Roberts, 1950; Crafts, 1951). Variation in results depending on form of virus suspension employed and/or type of plant tested may have been related to concentrations of virus to which plant roots were exposed, and/or to qualitative differences in virus preparations. Processes associated with normal root growth, as for example, emergence and rupture of root hairs or lateral roots, may allow viruses or other macromolecules to enter internal root tissue. Whether viruses are translocat’ed to upper parts of roots or aerial portions of plants by simple capillary ascension through root interiors, or by more active transport mechanisms, is not known. Even with plant viruses special techniques are required to facilitate acropetal translocation (Bennett, 1940). Titmtions of plant extracts provided no evidence to suggest propagation of mouse encephaIomyelitis or poliomyelitis virus in plant tissue. Foods have been known to serve as natural vehicles for infection perorally by many microbiologic agents. Despite epidemiologic evidence to suggest many times that this mode for infection has operated in poliomyelitis, experimental attempts to provide supportive evidence for a relationship between the food ingested and onset of infection have failed or proved equivocal. These attempts have covered a wide range of experimental studies. Toomey and his associates (1943) failed during a poliomyelitis outbreak to demonstrate poliovirus in washings of fresh

ABSORPTION

.4ND

TRANSLOCATION

OF MAMMALIAN

VIRUSES.

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fruits, vegetables, and other materials assayed for virus in cotton rats, monkeys, and other animals. Gebhardt and Woodie (1948) attempted unsuccessfully to recover virus from washings of fruit handled by a peddler known to be a fecal poliovirus carrier. Toomey and Corum (1943) failed to recover poliovirus from tomato plants which had been inoculated by inject’ion of poliovirus with syringe and needle into the plant stem or by rubbing virus suspension into young leaves. Moryzki and associates (1952) &ted in an abstract without supportive evidence that t,ype 2 poliovirus penetrated undamaged roots to survive for 4 days in root tissue. Moryzki made this claim without description of bhe experimental conditions and without the supportive evidence that is requisite for proper evaluation and confirmation by other investigators. In retrospect it is not unlikely that loss of virus in t,he process of ultrafiltration may be held responsible for failure of virus recovery in the studies reported by Toomey et al. and by Gebhardt. Gear and Measroch (1949) and Gear (1952) reported unsuccessful attempts to recover poliovirus (a) from common garden vegetables grown on a sewage farm irrignt’ed wit,h the effluent from a sewage bank known to contain poliovirus or (b) from the fruit of tomato plants grown in hydroponic solution containing poliovirus. The findings reported herein and the findings reported by Gear and Mensroch are not necessarily in disagreement since it was shown in this laborat,ory (1948) t,hat rapid adsorption of free virus by soil makes it unavailable for plant absorption. In summary, virus can be absorbed by pla,nt roots under adequate conditions, but translocat.ion is uncommon. From these findings, it is unlikely that plants or plant’ fruits serve as a reservoir and/or carrier of poliovirus. REF15RENCIB I%AWDES, F. C:., and PIRIE, W. W. (1951). Some effects of freezing in the leaf and of citrate in ktro on the infectivity of a tobacco necrosis virus. J. Gen. hJicrobiol. 4, 482-492. RENFETT, C. W. (1940). Relation of food tjransloration to movement of virus of t.obawo mosaic. J. :I qri. 12esenwh 60, 361-390. CAPOOR, S. 1’. (1949). Movement of tobacco mosaic virus and potato virus S through tomato plants. rlnn. Appl. Biol. 36, 307-318. CHANTRILL, B. H., COULTHARD, C. E., DICKENSOX, Id., INKLEY, G. W., MORRIS, W., and PYLE, A. H. (1952). The action of plant extracts on a bacteriophage of Pseudomonns pyocyansa and on infllwnza A virus. J. Gen. Microbial. 6, 74-8-t. CHESTER, K. S. (1937). A critique of plant serology. Quart. Rev. RioE. 12, 29-1-321. CRAFTS, A. S. (1951). Movement of assimilates, viruses, growth regulators, and chemical indicators in plants. Botan. RCZJ.17, 203-253.

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DANIELS, J. B., PAPPENHEIMER, A. M., and RICHARDSON, S. (1952). Observations on encephalomyelitis of mice (DA strain). J. Exptl. Med. 96,517-530. DEAN, J. D. (1951). Mouse encephalomyelitis. Immunologic st,udies of a non-infected colony. J. Immunol. 66, 347-359. FAJARDO, T. G. (1930). Studies on the mosaic disease of the bean (Phnseolus vulgaris L). Phytopathology 20,469494. FULTON, R. W. (1941). The behavior of certain viruses in plant roots. Phytopathology 31,57&598. GEAR, J. H. S. (1952). Ecology of poliomyelit,is. In Poliomyelitis: Papers and Discussions Presented at the Second International Poliomyelitis Conference, pp. 343-354. Lippincott, Philadelphia. GEAR, J. H. S., and MEASROCH, V. (1949). Poliomyelitis and sewage. Public Health Johannesburg 13, 37-39. GEBHARDT, L. P., and WOOUIE, J. D. (1948). Possible dissemination of poliomyelitis virus by carriers. Am. J. Diseases Children 76, 192-193. MORYZKI, J., KAWESKI, A., and KORNOWICZ, W. (1952). Experimental investigations concerning the epidemiological role of green vegetables in poliomyelitis. Bull. State Inst. Marine and Trop. Med. Gddnsk, Poland 4, 131-134. MURPHY, JR., W. H., EYLAR, 0. R., SCHMIDT, E. L., and SYVERTON, J. T. (1958). Absorption and translocation of mammalian viruses by plants. I. Survival of mouse encephalomyelitis and poliomyelitis viruses in soil and plant root environment. virology 6, 612-622. ROBERTS, F. M. (1950). The infection of plant,s by viruses through roots. Ann. Appl. Biol. 36,418428. SALK, J. E., LEWIS, L. J., YOUNGNER, J. S., and BENNETT, B. L. (1951). The use of adjuvants to facilitate studies on the immunologic classification of poliomyclitis viruses. Am. J. Hyg. 64, 157-173. SKARNES, R. C. (1952). Studies upon the persistence of animal viruses in soil and their uptake by higher plants. M.S. Thesis, University of Minnesota, Minneapolis, Minnesota. SYVERTON, J. T., SCHERER, W. F., and ELLWOOD, P. M. (1954). Studies on the propagation in. vitro of poliomyelitis viruses. V. The application of strain HeLa human epithelial cells for isolation and typing. J. Lab. Clin. Med. 43, 28&302. TOOMEY, J. A., and CORUM, C. J. (1943). Attempts to acclimate poliomyelitis virus to tomato plants. Proc. Sot. Ezptl. Biol. Med. 64, 10-11. TOOMEY, J. A., TAKAC~, W. S., and TISCHER, L. A. (1943). Attempts to recover poliomyelitis virus from fruit, well water, chicken cords and dog stools. J. Pediat. 23, 168-171. WEIL, M. L., WARREN, J., BREESE, S. S., JR., Russ, S. B., and JEFFRIES, H. (1952). Separation of encephalomyocarditis virus from tissue components by means of protamine precipitation and enzymic digestion. J. Bacterial. 63,99-105.