- Email: [email protected]
Aphid honeydew as inoculum for the injection of pea aphids with pea-enation mosaic virus
472
DISCUSSION AND PRELIMINARY REPORTS
FIG. 1 . Osmiophilic particles in the salivary gland in cross and longitudinal orientations .
Aphid Honeydew as Inoculum for the Injection of Pea Aphids with Pea-Enation Mosaic Virus'
Studies on the persistent aphid-borne viruses are limited by the lack of simple bioassays . Since most of these viruses arc not juice inoculable, vectors are required for the bioassay of various virus-containing extracts . Carter (3) demonstrated that the leafhopper Circulifer tenellus (Baker) transmitted curly top virus after feeding through a membrane into solutions containing virus . Storey (17) repeated this technique with maize streak virus and in addition showed that leafhoppers transmitted virus after being injected with infectious plant, extracts (16) . The needle injection technique has become rather commonly used for leafhopper-borne viruses . ' Supported in part by a grant from the United States Public Health Service, AT 03497 .
of
the leafhopper
Endria in ica
Say . Particles
These two bioassay techniques have been recently applied to the aphid-borne persistent viruses . Barley yellow dwarf virus has been transmitted by the English grain aphid, Macrosiphuin granarium (Kirby), and by the apple grain aphid Rhopalosiphum pacli (L .) after feeding on liquid extracts from diseased plants (12, 13) . Although this is the only published example of successful acquisition of a persistent aphid-borne virus by the artificial feeding technique, perhaps the recent successes of 2\-littler and Dadd (9) and of Auclair and Cartier (2) in the artificial rearing of aphids will encourage further trials . More success has been obtained with needle inoculation of aphids, since four viruses have been transmitted by this method . Transmission of potato leaf roll virus has been reported after injection of green peach aphids. Myzus persicae (Sulzer),
DISCUSSION AND PRELINIINAR1 REPORTS with infectious hemolymph or extracts from viruliferous aphids (4, 5, 7, 15) . Limited success with beet yellow-net virus was reported by Harrison (5) . Although Heinze (6) failed to obtain transmission of pea-enation mosaic virus by injection of hemolymph from viruliferous pea aphids, Acyrthosiphou pisum (Harris), later work by Schmidt (14) and by Nault et al . (11) showed that limited transmission could be obtained . Mueller and Rochow (10) reported transmission of barley yellow dwarf virus by both the English grain aphid and the apple grain aphid after injection of inoculuin from plant or aphid extracts or with hemolymph from viruliferous aphids . Certain studies on vector-virus relationships, e .g ., the relationship between latent period and/or retention to virus dosage, can only be done by a bioassay technique such as artificial feeding or injection . Recently we have spent considerable time investigating some of these vector-virus relationships of the pea aphid and pea-enation mosaic virus (18) ; in order to utilize the injection technique in further studies, we have searched for a suitable source of inoculum . The plants for the experiments were sweetpea, Lathyrus odoratus L., raised from seed in individual 3-inch plastic pots filled with pasteurized soil . The test plants were generally used about 7 days after planting, and virus source plants were used within 2 weeks after the appearance of symptoms . The virus was a strain of pea-enation mosaic, virus isolated from a sweetpea plant growing in a garden in Berkeley, California . The virus originally was kept in the greenhouse in bur clover, Medicago hispida Gaertn ., but for the last two years it has been subinoculated into sweetpea . Nymphs and apterae from a clonal line of the pea aphid vector were raised in colonies on sweetpea, and the preliminary experimental work reported here was done in a 6-month period beginning in May, 1964 . The aphids injected were mainly late instar nymphs, but, some adults were also used . Aphids were anesthetized with moistened CO 2 , placed on their sides on. a piece of rough cardboard on the stage of a stereomicroscope, and injected laterally near the corniele by means of a twice-pulled glass
473
needle? The injection needle was connected to an air supply by rubber tubing and a glass T-joint ; closure of one end of the '1'-joint forced inoculum out of the needle into the aphid . Plant extracts were prepared by grinding an equal (w/v) amount of diseased tissue and solvent containing 0 .05 M K2HPO4 and 0 .01 M Na 2SO 3 in a mortar arid sgeezing it through two thicknesses of cheesecloth . The fluid was then centrifuged at 1600 g for 10 minutes, and the supernatant was used for the injections . The plant extracts were drawn up into the needle by connecting it to a small vacuum pump? The amount of liquid drawn into the needle was sufficient to inject two or three dozen aphids . In the hemolymph tests, the donor aphids were anesthetized and placed on rough cardboard oil the stage, of the stereomicroscope . A leg was cut off, and the hemolymph that oozed from the wound was collected in an injection needle by capillary action . This material was immediately injected into a recipient aphid . In the honeydew tests, the plants on which the donor aphids were feeding were caged with glass vials . Small droplets of honeydew ejected to the sides of the vials by the aphids were picked up in the needles by capillary action . One droplet from an adult donor aphid was sufficient to inject an average of 5 aphids . If the honeydew collections were to be pooled, successive droplets were collected in a finely drawn glass tube and then mixed by blowing the liquid out and collecting it again in the tube . The injection needles were then filled from this pooled honeydew . Injected aphids in all experiments were caged on healthy sweetpea test plants in cellulose nitrate cages, and the insects were serially transferred to additional test plants at varying intervals for several days . The test plants were kept in a Sherer-Gillett model CEL 512-317 plant growth chamber 2 The injection needles were pulled automatically with a device designed and constructed by Professor R . Craig, Division of Entomology and Acarology, University of California, Berkeley . 3 Vacuum Tweezer System, model V-100, Ultrasonic Laboratories, Inc ., 1780 St . Georges Avenue, Rahway, New Jersey .
4 74
DISCO~4STON Ann PHELIMeNAttY REPORTS
during the inoculation access period . The chamber was programmed to have a temperature of 20° C with continuous light . At the end of the inoculation access period, the aphids were removed from the plants, and the plants were fumigated with nicotine before being placed in the greenhouse to complete the incubation period . A low rate of transmission was obtained with aphids injected with crude plant extracts . Of 49 aphids injected and tested singly, only 3 transmitted virus . Only eight of 72 recipient aphids transmitted virus after each had been injected with hemolymph from a viruliferous donor aphid . With pooled hemolymph, collected in a single needle from the wounds of several aphids that had had access to a virus-infected plant, 5 of 60 injected aphids transmitted virus . A high rate of transmission was achieved by injecting honeydew excreted by aphids feeding on virus-infected plants . In the initial trials, 24 of 50 test plants became infected after each had been fed upon by 2 injected aphids . This corresponds to an expected transmission rate of approximately 28 per cent with single insects . Groups of 4 and 5 injected aphids per plant transmitted to 18 of 20 test plants, a rate of transmission too high to be reliably expressed in terms of single-insect expectancy . To test how soon infectious honeydew would he excreted, a group of aphids was transferred from healthy plants to a series of diseased plants, and the ejected droplets of honeydew were picked up in needles and injected into nonviruliferous nymphs. The honeydew was excreted and assayed over a period of time varying from 15 minutes to approximately 7 hours . Thirty-six such droplets were tested of which 11 proved to be infectious . The minimum access period after which an infectious honeydew droplet was excreted was approximately 1 .5 hours . An experiment was done to determine how long aphids that have left a diseased plant would continue to excrete infectious honeydew Thirty nonviruliferous aphids were allowed access to virus source plants for 12 hours at 20 °C . The aphids were then trans-
ferred to 30 healthy sweetpea seedlings, one aphid per plant, and every 24 hours the insects were moved to new test plants for a total of 7 days . The first honeydew droplets excreted by each aphid upon leaving the virus source plants were assayed, and 19 aphids were found to have excreted infectious honeydew . But when the honeydew excreted by the donor aphids on the 2nd, 3rd, and the 7th day of feeding on healthy plants was assayed, only 2, 1, and 0 aphids were found to have excreted infectious honeydew on these days, respectively . Twenty-three of the 30 aphids acquired virus during the 12-hour acquisition access period, but only 1S of these excreted infectious honeydew . In one case a honeydew droplet was infectious although the aphid from which it came failed to transmit virus to any of the 7 test plants upon which it subsequently fed . The honeydew excreted by pea aphid apterae feeding on sweetpea plants is slightly alkaline, as tested by pH paper . This is close to the value of 7 .8-8 .0 given by Auclair (1) for pea aphids feeding on pea, Pisum satiwum L . Limited dilution tests have indicated that honeydew can be diluted to 1O with either distilled water or 0 .1 Bf phosphate buffer, pH 7 .1, and still remain infectious to inoculated aphids . Of all the sources of inoculum we have tested, viz., plant extracts, hemolymph from viruliferous aphids, and honeydew excreted by aphids feeding on virus-infected plants, the last-mentioned appears to be the most promising . Honeydew of the pea aphid is readily collected, and a single droplet from an adult is sufficient to inoculate several insects . Honeydew is qualitatively similar in cornposition to the phloem sap of the plant oil which the aphids feed (8) and from which the pea-enation mosaic virus is presumably acquired . Honeydew contains a mixture of excess carbohydrates, amino acids, water, and other phloem products, with some additional metabolic and excretory materials from the, aphids (19), but apparently lacks any significant amount of protein (1) . The honeydew excreted by the pea aphid is clear
DISCUSSION AND PRELIMINARY REPORTS and, in spite of the obvious quantitative limitations, should serve as a good medium from which to concentrate or purify virus . Perhaps virus particles could be visualized by electron microscopic examination of honeydew . Preliminary work has also indicated the suitability of the injection technique using virus-bearing honeydew for the determination of pH stability and dilution studies . It, should prove equally adaptable for determining other properties of viruses . REFERENCES 1.
AucLAIR,
J . L., Ann . Rev . Ento,nol . 8, 439-490
(1963) . 2.
J . L ., and CARTIER, J . J . . Science 142, 1068-1069 (1963) . 3 . CARTER, W ., J. Agr . Res . 34, 449-451 (1927) . 4 . DAY, M . F ., Australian J. Biol. Sci . 8, 498-513 (1955) . 5 . HARRISON, B . D ., Virology 6, 265-277 (1958) . 6 . HEINZE, Ii ., L Evan . Entomol . 48, 751 (1955) . 7 . HEINZE, K ., Phytopathol . Z . 2 .5, 103-108 (1955) . 8 . MITTnER, T . E ., J . Exptl . Biel . 35, 74-84 (1958) . 9 . MITTLER, T . E ., and Dens, It. H ., Nature 195, 404 (1962) . 10 . MUELLER, W . C ., and Rocimw, W . F ., Virology 14, 253-258 (1961) . 11 . NAU,LT, L . R., GYmsco, G . G ., and ltocnow, W . F ., Phytopathology 54, 1269-1272 (1964) . 12 . PAN, E-WA, and Rocnow, W . F . (Abstract), Phytopathology 53, 351 (1963) . 13 . RocuOw, W . F ., Virology 12, 223-232 (1960) . 14 . SOIIMIDT, H . B ., Biol . Zentr . 78, 889-936 (1959) . 15 . STEOWEE, .D . . and PONSON, M . B . . Entomol . Exptl . Appl . 1, 291-300 (1958) . 16 . STOREY, H . H ., Proc . Roy . Soc . B113, 463-485 (1933) . 17 . STOREY, H H ., Proc . Roy. Sac . B127, 526-543 (1939) . 18 . SYLVESTER, E . S ., Virology, 25, 62-67 (1965) . 19 . WATERROUSE, D . F ., Ann . Rev . Entomol . 2, 1-18 (1957) . JEAN RICIIARDSON 4 E . S . SYLvE,,rER Division of Entomology and Acarology AUCLAIR,
California. California Accepted January 11, 1965
University of Berkeley,
' Laboratory Technician and Professor, respectively .
475
The Development of Ultraviolet-Irradiation Resistance by Poliovirus Infective Centers and Its Inhibition by Guanidine) Guanidine, a selective inhibitor of picornavirus reproduction, has been shown to inhibit, the synthesis of viral RNA polymerase and of viral RNA [cf . review by Tamm and Eggers (1)] . The problem as to when in the multiplication cycle of poliovirus type 1 the synthesis of virus-directed macromolecules begins has been investigated in kinetic experiments in which guanidine was removed by washing the cultures at different times after infection . The results indicated that the guanidine-inhibitable period begins about 1 hour after virus inoculation (2), but biologic and isotope data showed that guanidine is not readily removed from cell cultures by washing with guanidine-fret medium (2) . The possibility could therefore not be excluded that the guanidine-sensitive process actually begins somewhat later than inferred from the kinetic experiments . A different approach to the problem of the early events in poliovirus replication is available in the Luria-Latarjet type of experiment (3) as applied to the poliovirus system (4) . In the present study, the acquisition of ultraviolet (UV)-irradiation resistance by infective centers was followed in the presence or absence of guanidine . For comparison, infected cells treated with puromycin were also examined . The removal of the compound from the cells is not a necessary part of this experimental design . The results of a typical experiment with guanidine-sensitive poliovilus 1 (13runenders) are presented in rig . 1 . In addition, the inactivation kinetics of the free virus are shown . UV-irradiation of infectivc centers was performed after a total incubation period at 37° of 50, 110, and 180 minutes following virus inoculation . The slope of the inactivation curve of the infective centers from untreated cultures, determined at 50 minutes following infection, is slightly less steep than that of free virus, in agreement with results obtained by other investigators (8. 9) . Not illustrated in the graph is the I Aided by a grant from The National Foundation .
DISCUSSION AND PRELIMINARY REPORTS
FIG. 1 . Osmiophilic particles in the salivary gland in cross and longitudinal orientations .
Aphid Honeydew as Inoculum for the Injection of Pea Aphids with Pea-Enation Mosaic Virus'
Studies on the persistent aphid-borne viruses are limited by the lack of simple bioassays . Since most of these viruses arc not juice inoculable, vectors are required for the bioassay of various virus-containing extracts . Carter (3) demonstrated that the leafhopper Circulifer tenellus (Baker) transmitted curly top virus after feeding through a membrane into solutions containing virus . Storey (17) repeated this technique with maize streak virus and in addition showed that leafhoppers transmitted virus after being injected with infectious plant, extracts (16) . The needle injection technique has become rather commonly used for leafhopper-borne viruses . ' Supported in part by a grant from the United States Public Health Service, AT 03497 .
of
the leafhopper
Endria in ica
Say . Particles
These two bioassay techniques have been recently applied to the aphid-borne persistent viruses . Barley yellow dwarf virus has been transmitted by the English grain aphid, Macrosiphuin granarium (Kirby), and by the apple grain aphid Rhopalosiphum pacli (L .) after feeding on liquid extracts from diseased plants (12, 13) . Although this is the only published example of successful acquisition of a persistent aphid-borne virus by the artificial feeding technique, perhaps the recent successes of 2\-littler and Dadd (9) and of Auclair and Cartier (2) in the artificial rearing of aphids will encourage further trials . More success has been obtained with needle inoculation of aphids, since four viruses have been transmitted by this method . Transmission of potato leaf roll virus has been reported after injection of green peach aphids. Myzus persicae (Sulzer),
DISCUSSION AND PRELINIINAR1 REPORTS with infectious hemolymph or extracts from viruliferous aphids (4, 5, 7, 15) . Limited success with beet yellow-net virus was reported by Harrison (5) . Although Heinze (6) failed to obtain transmission of pea-enation mosaic virus by injection of hemolymph from viruliferous pea aphids, Acyrthosiphou pisum (Harris), later work by Schmidt (14) and by Nault et al . (11) showed that limited transmission could be obtained . Mueller and Rochow (10) reported transmission of barley yellow dwarf virus by both the English grain aphid and the apple grain aphid after injection of inoculuin from plant or aphid extracts or with hemolymph from viruliferous aphids . Certain studies on vector-virus relationships, e .g ., the relationship between latent period and/or retention to virus dosage, can only be done by a bioassay technique such as artificial feeding or injection . Recently we have spent considerable time investigating some of these vector-virus relationships of the pea aphid and pea-enation mosaic virus (18) ; in order to utilize the injection technique in further studies, we have searched for a suitable source of inoculum . The plants for the experiments were sweetpea, Lathyrus odoratus L., raised from seed in individual 3-inch plastic pots filled with pasteurized soil . The test plants were generally used about 7 days after planting, and virus source plants were used within 2 weeks after the appearance of symptoms . The virus was a strain of pea-enation mosaic, virus isolated from a sweetpea plant growing in a garden in Berkeley, California . The virus originally was kept in the greenhouse in bur clover, Medicago hispida Gaertn ., but for the last two years it has been subinoculated into sweetpea . Nymphs and apterae from a clonal line of the pea aphid vector were raised in colonies on sweetpea, and the preliminary experimental work reported here was done in a 6-month period beginning in May, 1964 . The aphids injected were mainly late instar nymphs, but, some adults were also used . Aphids were anesthetized with moistened CO 2 , placed on their sides on. a piece of rough cardboard on the stage of a stereomicroscope, and injected laterally near the corniele by means of a twice-pulled glass
473
needle? The injection needle was connected to an air supply by rubber tubing and a glass T-joint ; closure of one end of the '1'-joint forced inoculum out of the needle into the aphid . Plant extracts were prepared by grinding an equal (w/v) amount of diseased tissue and solvent containing 0 .05 M K2HPO4 and 0 .01 M Na 2SO 3 in a mortar arid sgeezing it through two thicknesses of cheesecloth . The fluid was then centrifuged at 1600 g for 10 minutes, and the supernatant was used for the injections . The plant extracts were drawn up into the needle by connecting it to a small vacuum pump? The amount of liquid drawn into the needle was sufficient to inject two or three dozen aphids . In the hemolymph tests, the donor aphids were anesthetized and placed on rough cardboard oil the stage, of the stereomicroscope . A leg was cut off, and the hemolymph that oozed from the wound was collected in an injection needle by capillary action . This material was immediately injected into a recipient aphid . In the honeydew tests, the plants on which the donor aphids were feeding were caged with glass vials . Small droplets of honeydew ejected to the sides of the vials by the aphids were picked up in the needles by capillary action . One droplet from an adult donor aphid was sufficient to inject an average of 5 aphids . If the honeydew collections were to be pooled, successive droplets were collected in a finely drawn glass tube and then mixed by blowing the liquid out and collecting it again in the tube . The injection needles were then filled from this pooled honeydew . Injected aphids in all experiments were caged on healthy sweetpea test plants in cellulose nitrate cages, and the insects were serially transferred to additional test plants at varying intervals for several days . The test plants were kept in a Sherer-Gillett model CEL 512-317 plant growth chamber 2 The injection needles were pulled automatically with a device designed and constructed by Professor R . Craig, Division of Entomology and Acarology, University of California, Berkeley . 3 Vacuum Tweezer System, model V-100, Ultrasonic Laboratories, Inc ., 1780 St . Georges Avenue, Rahway, New Jersey .
4 74
DISCO~4STON Ann PHELIMeNAttY REPORTS
during the inoculation access period . The chamber was programmed to have a temperature of 20° C with continuous light . At the end of the inoculation access period, the aphids were removed from the plants, and the plants were fumigated with nicotine before being placed in the greenhouse to complete the incubation period . A low rate of transmission was obtained with aphids injected with crude plant extracts . Of 49 aphids injected and tested singly, only 3 transmitted virus . Only eight of 72 recipient aphids transmitted virus after each had been injected with hemolymph from a viruliferous donor aphid . With pooled hemolymph, collected in a single needle from the wounds of several aphids that had had access to a virus-infected plant, 5 of 60 injected aphids transmitted virus . A high rate of transmission was achieved by injecting honeydew excreted by aphids feeding on virus-infected plants . In the initial trials, 24 of 50 test plants became infected after each had been fed upon by 2 injected aphids . This corresponds to an expected transmission rate of approximately 28 per cent with single insects . Groups of 4 and 5 injected aphids per plant transmitted to 18 of 20 test plants, a rate of transmission too high to be reliably expressed in terms of single-insect expectancy . To test how soon infectious honeydew would he excreted, a group of aphids was transferred from healthy plants to a series of diseased plants, and the ejected droplets of honeydew were picked up in needles and injected into nonviruliferous nymphs. The honeydew was excreted and assayed over a period of time varying from 15 minutes to approximately 7 hours . Thirty-six such droplets were tested of which 11 proved to be infectious . The minimum access period after which an infectious honeydew droplet was excreted was approximately 1 .5 hours . An experiment was done to determine how long aphids that have left a diseased plant would continue to excrete infectious honeydew Thirty nonviruliferous aphids were allowed access to virus source plants for 12 hours at 20 °C . The aphids were then trans-
ferred to 30 healthy sweetpea seedlings, one aphid per plant, and every 24 hours the insects were moved to new test plants for a total of 7 days . The first honeydew droplets excreted by each aphid upon leaving the virus source plants were assayed, and 19 aphids were found to have excreted infectious honeydew . But when the honeydew excreted by the donor aphids on the 2nd, 3rd, and the 7th day of feeding on healthy plants was assayed, only 2, 1, and 0 aphids were found to have excreted infectious honeydew on these days, respectively . Twenty-three of the 30 aphids acquired virus during the 12-hour acquisition access period, but only 1S of these excreted infectious honeydew . In one case a honeydew droplet was infectious although the aphid from which it came failed to transmit virus to any of the 7 test plants upon which it subsequently fed . The honeydew excreted by pea aphid apterae feeding on sweetpea plants is slightly alkaline, as tested by pH paper . This is close to the value of 7 .8-8 .0 given by Auclair (1) for pea aphids feeding on pea, Pisum satiwum L . Limited dilution tests have indicated that honeydew can be diluted to 1O with either distilled water or 0 .1 Bf phosphate buffer, pH 7 .1, and still remain infectious to inoculated aphids . Of all the sources of inoculum we have tested, viz., plant extracts, hemolymph from viruliferous aphids, and honeydew excreted by aphids feeding on virus-infected plants, the last-mentioned appears to be the most promising . Honeydew of the pea aphid is readily collected, and a single droplet from an adult is sufficient to inoculate several insects . Honeydew is qualitatively similar in cornposition to the phloem sap of the plant oil which the aphids feed (8) and from which the pea-enation mosaic virus is presumably acquired . Honeydew contains a mixture of excess carbohydrates, amino acids, water, and other phloem products, with some additional metabolic and excretory materials from the, aphids (19), but apparently lacks any significant amount of protein (1) . The honeydew excreted by the pea aphid is clear
DISCUSSION AND PRELIMINARY REPORTS and, in spite of the obvious quantitative limitations, should serve as a good medium from which to concentrate or purify virus . Perhaps virus particles could be visualized by electron microscopic examination of honeydew . Preliminary work has also indicated the suitability of the injection technique using virus-bearing honeydew for the determination of pH stability and dilution studies . It, should prove equally adaptable for determining other properties of viruses . REFERENCES 1.
AucLAIR,
J . L., Ann . Rev . Ento,nol . 8, 439-490
(1963) . 2.
J . L ., and CARTIER, J . J . . Science 142, 1068-1069 (1963) . 3 . CARTER, W ., J. Agr . Res . 34, 449-451 (1927) . 4 . DAY, M . F ., Australian J. Biol. Sci . 8, 498-513 (1955) . 5 . HARRISON, B . D ., Virology 6, 265-277 (1958) . 6 . HEINZE, Ii ., L Evan . Entomol . 48, 751 (1955) . 7 . HEINZE, K ., Phytopathol . Z . 2 .5, 103-108 (1955) . 8 . MITTnER, T . E ., J . Exptl . Biel . 35, 74-84 (1958) . 9 . MITTLER, T . E ., and Dens, It. H ., Nature 195, 404 (1962) . 10 . MUELLER, W . C ., and Rocimw, W . F ., Virology 14, 253-258 (1961) . 11 . NAU,LT, L . R., GYmsco, G . G ., and ltocnow, W . F ., Phytopathology 54, 1269-1272 (1964) . 12 . PAN, E-WA, and Rocnow, W . F . (Abstract), Phytopathology 53, 351 (1963) . 13 . RocuOw, W . F ., Virology 12, 223-232 (1960) . 14 . SOIIMIDT, H . B ., Biol . Zentr . 78, 889-936 (1959) . 15 . STEOWEE, .D . . and PONSON, M . B . . Entomol . Exptl . Appl . 1, 291-300 (1958) . 16 . STOREY, H . H ., Proc . Roy . Soc . B113, 463-485 (1933) . 17 . STOREY, H H ., Proc . Roy. Sac . B127, 526-543 (1939) . 18 . SYLVESTER, E . S ., Virology, 25, 62-67 (1965) . 19 . WATERROUSE, D . F ., Ann . Rev . Entomol . 2, 1-18 (1957) . JEAN RICIIARDSON 4 E . S . SYLvE,,rER Division of Entomology and Acarology AUCLAIR,
California. California Accepted January 11, 1965
University of Berkeley,
' Laboratory Technician and Professor, respectively .
475
The Development of Ultraviolet-Irradiation Resistance by Poliovirus Infective Centers and Its Inhibition by Guanidine) Guanidine, a selective inhibitor of picornavirus reproduction, has been shown to inhibit, the synthesis of viral RNA polymerase and of viral RNA [cf . review by Tamm and Eggers (1)] . The problem as to when in the multiplication cycle of poliovirus type 1 the synthesis of virus-directed macromolecules begins has been investigated in kinetic experiments in which guanidine was removed by washing the cultures at different times after infection . The results indicated that the guanidine-inhibitable period begins about 1 hour after virus inoculation (2), but biologic and isotope data showed that guanidine is not readily removed from cell cultures by washing with guanidine-fret medium (2) . The possibility could therefore not be excluded that the guanidine-sensitive process actually begins somewhat later than inferred from the kinetic experiments . A different approach to the problem of the early events in poliovirus replication is available in the Luria-Latarjet type of experiment (3) as applied to the poliovirus system (4) . In the present study, the acquisition of ultraviolet (UV)-irradiation resistance by infective centers was followed in the presence or absence of guanidine . For comparison, infected cells treated with puromycin were also examined . The removal of the compound from the cells is not a necessary part of this experimental design . The results of a typical experiment with guanidine-sensitive poliovilus 1 (13runenders) are presented in rig . 1 . In addition, the inactivation kinetics of the free virus are shown . UV-irradiation of infectivc centers was performed after a total incubation period at 37° of 50, 110, and 180 minutes following virus inoculation . The slope of the inactivation curve of the infective centers from untreated cultures, determined at 50 minutes following infection, is slightly less steep than that of free virus, in agreement with results obtained by other investigators (8. 9) . Not illustrated in the graph is the I Aided by a grant from The National Foundation .