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Detection of nuclear polyhedrosis virus infection in Heliothis spp. by agar gel double diffusion
JOURNAL
OF INVERTEBRATE
PATHOLOGY
Detection in Heliothis
26, 309-312 (1975)
of Nuclear
Polyhedrosis
Virus lnfectiqn
spp. by Agar Gel Double Diffusion
S. Y. YOUNG,
W. C. YEARIAN,
AND
H. A. SCOTT
Virology and Biocontrol Laboratory, University Farms, University of Arkansas, Fayetteville, Arkansas 72701 Received January 15, 1975 Nuclear Polyhedrosis virus infection was detected in single Heliothis zea larvae by the agar gel double diffusion technique using antiserum to alkali-solubilized polyhedra. Virus bands were observed in 2nd-5th stadium larvae following homogenization in 0.1 M Na*CO,-0.05 M NaCI, pH 11.0, in approximately one-half the time necessary for mortality to occur. In a field test, virus infection was detected by this method as early as 3 days after virus treatment.
INTRODUCTION Techniques for determining the incidence of virus infection in insect populations are needed for rapid assessment of efficacy of virus treatments and for monitoring viruses in the environment. Serological techniques are routinely used to monitor plant (Corbett and Sisler, 1964) and vertebrate viruses (Debre and Celers, 1970) in host populations and offer potential for assessing the incidence of virus in an insect population. Bailey (pers. comm.) has used immunodiffusion tests to detect chronic and acute bee paralysis viruses in the honeybee, Apis mellifera.
Although several serological investigations of nuclear polyhedrosis viruses (NPV) have been reported (Krywienczyk, 1962; Krywienczyk and Bergold, 1960a, 1960b, 1961; Cunningham, 1968; Scott and Young, 1973), little attention has been given to application of this information for monitoring the incidence of virus infection in insect populations. Young and Johnson (1972) showed that antibodies to alkali-solubilized polyhedra can be used to detect previral proteins in the fat body of NPV-infected larvae, thus suggesting the possibility of detecting viral infection prior to the formation of virions and polyhedra. Further, alkali solubi‘Published with the Approval of the Director, Arkansas Agricultural Experiment Station. 309 Copyright D 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
lization of polyhedra in the infected host would also serve as an additional source of inclusion body protein antigen. This investigation was conducted to assess the potential of the agar gel double diffusion technique (Ouchterlony, 1962) to monitor NPV infection in Heliothis spp. populations following field application of the virus. Prior to examining the incidence of virus infection in the field, studies were carried out in the laboratory to determine the sensitivity of this technique for detection of infection in different stage larvae. MATERIALS AND METHODS The isolate of Heliothis NPV used for antiserum production and laboratory studies was Biotrol VHZ, lot 17 (Nutrilite Products, Inc., Lakeview, California). Virus propagation and larval rearing conditions have been described previously (Young and Yearian, 1974). Partial purification of the virus, antiserum production to alkali-solubilized polyhedra in rabbits, and the immunodiffusion technique were essentially the same as described by Scott and Young (1973) for the Trichoplusia ni NPV. Rabbits were bled several times, and the antiserum used in this study had a titer of 1:64. Heliothis zea larvae were infected by feeding on diet surface treated with NPV. The dosage rates, which produced 100% mortality in 4-5 days, were as follows:
310
YOUNG ET AL.
neonate larvae, 7 polyhedral inclusion bodies (PIB)/mm2; l-day-old 1st instar, 70 PIB/ mm2; 2nd instar, 130 PIB/mm2; 3rd instar, 400 PIB/mm2; 4th instar, 7,000 PIB/mm2. Larvae were examined at 24, 48, and 72 hr post-infection. Individual larvae were homogenized in 0.1 M Na,CO,-0.05 ii4 NaCl, pH 11, and incubated at room temperature for 30 min. Homogenization was carried out in a conical centrifuge tube (12-ml capacity) with a glass rod. The volume of buffer in which larvae were homogenized was as follows: 1st and 2nd instar, 0.01 ml; 3rd instar, 0.02 ml; 4th instar, 0.04 ml; 5th instar, 0.06 ml. Approximately 50 larvae of each stage were used for each daily examination. Antibody reactive with normal larval antigens was removed by the intragel absorption technique (Crowle, 1961). The antiserum well was filled with homogenate from a healthy 4th instar and incubated for a minimum of 2 hr. Remaining fluid was removed, and antiserum was placed in the well. Surrounding antigen wells then were filled with homogenates of test larvae. The immunodiffusion plates were held in a saturated atmosphere containing 5% phenol as an antifungal agent. The plates were examined for band formation after 18-24 hr incubation. To make these bands permanent the gels were soaked 24 hr in 2% NaCl, dried, and stained with Amido Schwartz B. Field tests were conducted at Foreman, Arkansas, on Brycot 4 cotton. Plots of 0.2 A each were treated once with 40 larval equivalents (L. E.)/A and 160 L. E./A of Sandoz 240-070-l plus Shade, 1 lb/A and IMC 90016, a gustatory stimulant, 11.6 g/A (Sandoz-Wander, Inc., Homestead, Florida), respectively. A check plot was left untreated. Heliothis spp. population density in the plots at time of application was estimated at 15,000/A, and the species composition was approximately 60% H. zea and 40% H. virescens. Larvae of all ages were present at the time of application. From the 2nd through the 6th day following treatment, a minimum of 100 2nd-5th stage larvae were collected from the plots and
divided into two groups: small larvae (2nd3rd stages) and large larvae (4th-5th stages). Twenty-five larvae from each group were used for immunodiffusion tests. Gel plates were made, and wells punched in the laboratory. Gel was removed from the wells just prior to use in the field with a pipette attached to a Minivac vacuum pump. The remaining larvae in each group, up to 25, were held on diet and reared to pupation to determine the incidence of virus mortality. RESULTS AND DISCUSSION Antiserum prepared from alkali-digested polyhedra gave weak bands in immunodiffusion tests against homogenates of healthy bollworm larvae. Although virus specific bands were usually detectable in the presence of these bands, this was sometimes difficult, particularly with small larvae. Therefore, antibodies to healthy antigens were removed by intragel absorption with homogenates of healthy larvae (Fig. 1). These bands were probably due to insect tissue fragments in the NPV preparation used for antiserum production. In future studies better purification of polyhedra used for antiserum production is needed. Virus infection could not be detected at 24 hr in any age group, but bands were usually observed after 48 hr with homogenates of larvae in the 2nd-5th stages (Fig. 2). Virus infection was detected routinely in these larvae after 72 hr. Weak bands could often be detected in late 1st stage larvae at either 48 or 72 hr, but infection could not be detected routinely even after death. Preliminary studies were conducted to determine the conditions for homogenization of larvae that might increase the sensitivity of the test. We found that for early stage larvae it was necessary to homogenize the larvae in minimal quantities of buffer. To detect infection consistently in 2nd stage larvae at 48 hr post treatment, it was necessary to place the entire larval homogenate in the antigen well. Also, although viral infection could be detected in late stages of disease (72 hr) when larvae were homogenized in distilled water, more consistent results were
SEROLOGICAL
DETECTION
OF Heliothis NPV
311
pa‘: *01 FIG. I. Immunodiffusion of homogenates from healthy and diseased bollworm larvae. Fourth stage larvae were homogenized 2 days post infection with 7 x IO3 PIB/mmZ on diet. H, healthy; D, diseased; Pa, polyhedral antiserum; Pa-ab, polyhedral antiserum from which antibodies specific for normal larval antigens were removed by intragel absorption. FIG. 2. Immunodiffusion of bollworm larvae homogenates at 48 hr post infection. H, healthy; D, diseased; I, 2, 3,4,5, instars when tested; Pa-ab, polyhedral antiserum following intragel absorption with homogenates of healthy, 4th stage larvae.
obtained when alkali buffer was used and the homogenate incubated 30-60 min. When large larvae were homogenized in distilled water or low ionic strength buffer (0.01 M Na,CO,, pH 11 .O), considerable darkening of the area around the antigen well occurred, making it difficult to detect bands. Results of the field test using this serological technique for detection of larval TABLE 1 Frequency (“/c) of NPV Infection as Detected by Serology and Mortality (%) of Heliothis Larvae Following Field Application Treatment 40 LX Days post treatment 2 3 4 5 6
Ser. Mort. Ser. Mort. Ser. Mort. Ser. Mort. Ser. Mort.
160 L.E.
Larval stage
Larval stage
2-3
4-5
Av
2-3
4-5
Av
4.0 72.0 24.0 54.5 40.0 80.0 28.0 100.0 40.0 61.5
0.0 33.3 16.0 40.0 12.0 25.0 24.0 60.9 36.0 39.1
2.0 54.0 20.0 44.4 26.0 58.0 26.0 74.2 38.0 47.2
4.0 60.0 28.0 80.0 48.0 100.0 40.0 93.3 48.0 80.0
0.0 32.0 12.0 41.7 40.0 84.0 36.0 64.0 44.0 56.0
2.0 44.4 20.0 61.2 44.0 80.8 38.0 75.0 46.0 68.0
infection are given in Table 1. Virus infection was detected in a significant percentage of the population 3 days post treatment in both the 40 and 160 L.E. treated plots. A high incidence of infection was detected in larvae collected throughout the remainder of the test period (6 days). Infection was detected also in a greater percentage of small larvae than large larvae, particularly from the 40 L. E. treatment on the 3rd and 4th day. The mortality test generally showed a higher incidence of virus mortality than could be detected by the serological method. This was as expected since infected larvae in an early stage of disease when collected would not be detected by the serological technique but would eventually die from the disease. Serological detection gradually increased as the test progressed due to an increase in the number of larvae in the middle-to-late stages of disease at the later sample times. The results of this investigation demonstrated that immunodiffusion tests using antiserum to alkali-solubilized polyhedra can be used to detect virus infection routinely in 2nd instar and larger H. zea larvae during middle and late stages of
312
YOUNG ET AL.
disease. Since this technique is not sufficiently sensitive to detect viral infection during early stages of disease, it cannot be used to quantitate the incidence of infection within a population. This procedure shows promise, however, for utilization under field conditions for early predictions of trends in larval mortality. The advantages of this technique over more sensitive serological methods lie in its simplicity and the speed with which large samples can be tested. REFERENCES CORBETT, M. K., AND SISLER, H. D. 1964. “Plant Virology,” p. 527. University of Florida Press, Gainesville. CROWLE, A. J. 1961. Interpretation of immunodiffusion tests. Annu. Rev. Microbial. 14, 161-176. CUNNINGHAM, J. C. 1968. Serological and morphological identification of some nuclear-polyhedrosis and granulosis viruses. J. Invertebr. Pathol. 11, 132-141. DEBRE, R., AND CELERS,J. 1970. “Clinical Virology, the Evaluation and Management of Human Viral Infections,” p. 87 1. W. B. Saunders, Philadelphia, Pa.
KRYWIENCZYK,J. 1962. Immunoelectrophoretic studies of inclusion body proteins. J. Insect Pathol. 4, 185191. KRYWIENCZYK, J., AND BERGOLD, G. H. 1960a. Serological relationship of viruses from some lepidopterous and hymenopterous insects. Virology lo,3088315. KRYWIENCZYK, J., AND BERGOLD, G. H. 1960b. Serological relationship between inclusion body proteins of some Lepidoptera and Hymenoptera. J. Immunol.
84,404408.
KRYWIENCZYK, J., AND BERGOLD, G. H. 1961. Serological studies of inclusion-body proteins by agar diffusion techniques. J. fnsecf Pathol. 3, 15-24. OUCHTERLONY,0. 1962. Diffusion-in-gel methods for immunological analysis (II). Progr. AIIergy 6, 30154.
SCOTT, H. A., AND YOUNG, S. Y. 1973. Antigens associated with a nuclear polyhedrosis virus from cabbage looper larvae. J. Invertebr. Pathol. 21, 315317.
YOUNG, S. Y., AND JOHNSON, D. R. 1972. Nuclear polyhedrosis virus-specific soluble antigens in fat body of Trichoplusia ni larvae. J. Inverrebr. Pathol. 20,114-117.
YOUNG, S. Y., AND YEARIAN, W. C. 1974. Persistence of Heliothis NPV on foliage of cotton, soybean and tomato. Environ. Entomol. 3,253-255.
OF INVERTEBRATE
PATHOLOGY
Detection in Heliothis
26, 309-312 (1975)
of Nuclear
Polyhedrosis
Virus lnfectiqn
spp. by Agar Gel Double Diffusion
S. Y. YOUNG,
W. C. YEARIAN,
AND
H. A. SCOTT
Virology and Biocontrol Laboratory, University Farms, University of Arkansas, Fayetteville, Arkansas 72701 Received January 15, 1975 Nuclear Polyhedrosis virus infection was detected in single Heliothis zea larvae by the agar gel double diffusion technique using antiserum to alkali-solubilized polyhedra. Virus bands were observed in 2nd-5th stadium larvae following homogenization in 0.1 M Na*CO,-0.05 M NaCI, pH 11.0, in approximately one-half the time necessary for mortality to occur. In a field test, virus infection was detected by this method as early as 3 days after virus treatment.
INTRODUCTION Techniques for determining the incidence of virus infection in insect populations are needed for rapid assessment of efficacy of virus treatments and for monitoring viruses in the environment. Serological techniques are routinely used to monitor plant (Corbett and Sisler, 1964) and vertebrate viruses (Debre and Celers, 1970) in host populations and offer potential for assessing the incidence of virus in an insect population. Bailey (pers. comm.) has used immunodiffusion tests to detect chronic and acute bee paralysis viruses in the honeybee, Apis mellifera.
Although several serological investigations of nuclear polyhedrosis viruses (NPV) have been reported (Krywienczyk, 1962; Krywienczyk and Bergold, 1960a, 1960b, 1961; Cunningham, 1968; Scott and Young, 1973), little attention has been given to application of this information for monitoring the incidence of virus infection in insect populations. Young and Johnson (1972) showed that antibodies to alkali-solubilized polyhedra can be used to detect previral proteins in the fat body of NPV-infected larvae, thus suggesting the possibility of detecting viral infection prior to the formation of virions and polyhedra. Further, alkali solubi‘Published with the Approval of the Director, Arkansas Agricultural Experiment Station. 309 Copyright D 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
lization of polyhedra in the infected host would also serve as an additional source of inclusion body protein antigen. This investigation was conducted to assess the potential of the agar gel double diffusion technique (Ouchterlony, 1962) to monitor NPV infection in Heliothis spp. populations following field application of the virus. Prior to examining the incidence of virus infection in the field, studies were carried out in the laboratory to determine the sensitivity of this technique for detection of infection in different stage larvae. MATERIALS AND METHODS The isolate of Heliothis NPV used for antiserum production and laboratory studies was Biotrol VHZ, lot 17 (Nutrilite Products, Inc., Lakeview, California). Virus propagation and larval rearing conditions have been described previously (Young and Yearian, 1974). Partial purification of the virus, antiserum production to alkali-solubilized polyhedra in rabbits, and the immunodiffusion technique were essentially the same as described by Scott and Young (1973) for the Trichoplusia ni NPV. Rabbits were bled several times, and the antiserum used in this study had a titer of 1:64. Heliothis zea larvae were infected by feeding on diet surface treated with NPV. The dosage rates, which produced 100% mortality in 4-5 days, were as follows:
310
YOUNG ET AL.
neonate larvae, 7 polyhedral inclusion bodies (PIB)/mm2; l-day-old 1st instar, 70 PIB/ mm2; 2nd instar, 130 PIB/mm2; 3rd instar, 400 PIB/mm2; 4th instar, 7,000 PIB/mm2. Larvae were examined at 24, 48, and 72 hr post-infection. Individual larvae were homogenized in 0.1 M Na,CO,-0.05 ii4 NaCl, pH 11, and incubated at room temperature for 30 min. Homogenization was carried out in a conical centrifuge tube (12-ml capacity) with a glass rod. The volume of buffer in which larvae were homogenized was as follows: 1st and 2nd instar, 0.01 ml; 3rd instar, 0.02 ml; 4th instar, 0.04 ml; 5th instar, 0.06 ml. Approximately 50 larvae of each stage were used for each daily examination. Antibody reactive with normal larval antigens was removed by the intragel absorption technique (Crowle, 1961). The antiserum well was filled with homogenate from a healthy 4th instar and incubated for a minimum of 2 hr. Remaining fluid was removed, and antiserum was placed in the well. Surrounding antigen wells then were filled with homogenates of test larvae. The immunodiffusion plates were held in a saturated atmosphere containing 5% phenol as an antifungal agent. The plates were examined for band formation after 18-24 hr incubation. To make these bands permanent the gels were soaked 24 hr in 2% NaCl, dried, and stained with Amido Schwartz B. Field tests were conducted at Foreman, Arkansas, on Brycot 4 cotton. Plots of 0.2 A each were treated once with 40 larval equivalents (L. E.)/A and 160 L. E./A of Sandoz 240-070-l plus Shade, 1 lb/A and IMC 90016, a gustatory stimulant, 11.6 g/A (Sandoz-Wander, Inc., Homestead, Florida), respectively. A check plot was left untreated. Heliothis spp. population density in the plots at time of application was estimated at 15,000/A, and the species composition was approximately 60% H. zea and 40% H. virescens. Larvae of all ages were present at the time of application. From the 2nd through the 6th day following treatment, a minimum of 100 2nd-5th stage larvae were collected from the plots and
divided into two groups: small larvae (2nd3rd stages) and large larvae (4th-5th stages). Twenty-five larvae from each group were used for immunodiffusion tests. Gel plates were made, and wells punched in the laboratory. Gel was removed from the wells just prior to use in the field with a pipette attached to a Minivac vacuum pump. The remaining larvae in each group, up to 25, were held on diet and reared to pupation to determine the incidence of virus mortality. RESULTS AND DISCUSSION Antiserum prepared from alkali-digested polyhedra gave weak bands in immunodiffusion tests against homogenates of healthy bollworm larvae. Although virus specific bands were usually detectable in the presence of these bands, this was sometimes difficult, particularly with small larvae. Therefore, antibodies to healthy antigens were removed by intragel absorption with homogenates of healthy larvae (Fig. 1). These bands were probably due to insect tissue fragments in the NPV preparation used for antiserum production. In future studies better purification of polyhedra used for antiserum production is needed. Virus infection could not be detected at 24 hr in any age group, but bands were usually observed after 48 hr with homogenates of larvae in the 2nd-5th stages (Fig. 2). Virus infection was detected routinely in these larvae after 72 hr. Weak bands could often be detected in late 1st stage larvae at either 48 or 72 hr, but infection could not be detected routinely even after death. Preliminary studies were conducted to determine the conditions for homogenization of larvae that might increase the sensitivity of the test. We found that for early stage larvae it was necessary to homogenize the larvae in minimal quantities of buffer. To detect infection consistently in 2nd stage larvae at 48 hr post treatment, it was necessary to place the entire larval homogenate in the antigen well. Also, although viral infection could be detected in late stages of disease (72 hr) when larvae were homogenized in distilled water, more consistent results were
SEROLOGICAL
DETECTION
OF Heliothis NPV
311
pa‘: *01 FIG. I. Immunodiffusion of homogenates from healthy and diseased bollworm larvae. Fourth stage larvae were homogenized 2 days post infection with 7 x IO3 PIB/mmZ on diet. H, healthy; D, diseased; Pa, polyhedral antiserum; Pa-ab, polyhedral antiserum from which antibodies specific for normal larval antigens were removed by intragel absorption. FIG. 2. Immunodiffusion of bollworm larvae homogenates at 48 hr post infection. H, healthy; D, diseased; I, 2, 3,4,5, instars when tested; Pa-ab, polyhedral antiserum following intragel absorption with homogenates of healthy, 4th stage larvae.
obtained when alkali buffer was used and the homogenate incubated 30-60 min. When large larvae were homogenized in distilled water or low ionic strength buffer (0.01 M Na,CO,, pH 11 .O), considerable darkening of the area around the antigen well occurred, making it difficult to detect bands. Results of the field test using this serological technique for detection of larval TABLE 1 Frequency (“/c) of NPV Infection as Detected by Serology and Mortality (%) of Heliothis Larvae Following Field Application Treatment 40 LX Days post treatment 2 3 4 5 6
Ser. Mort. Ser. Mort. Ser. Mort. Ser. Mort. Ser. Mort.
160 L.E.
Larval stage
Larval stage
2-3
4-5
Av
2-3
4-5
Av
4.0 72.0 24.0 54.5 40.0 80.0 28.0 100.0 40.0 61.5
0.0 33.3 16.0 40.0 12.0 25.0 24.0 60.9 36.0 39.1
2.0 54.0 20.0 44.4 26.0 58.0 26.0 74.2 38.0 47.2
4.0 60.0 28.0 80.0 48.0 100.0 40.0 93.3 48.0 80.0
0.0 32.0 12.0 41.7 40.0 84.0 36.0 64.0 44.0 56.0
2.0 44.4 20.0 61.2 44.0 80.8 38.0 75.0 46.0 68.0
infection are given in Table 1. Virus infection was detected in a significant percentage of the population 3 days post treatment in both the 40 and 160 L.E. treated plots. A high incidence of infection was detected in larvae collected throughout the remainder of the test period (6 days). Infection was detected also in a greater percentage of small larvae than large larvae, particularly from the 40 L. E. treatment on the 3rd and 4th day. The mortality test generally showed a higher incidence of virus mortality than could be detected by the serological method. This was as expected since infected larvae in an early stage of disease when collected would not be detected by the serological technique but would eventually die from the disease. Serological detection gradually increased as the test progressed due to an increase in the number of larvae in the middle-to-late stages of disease at the later sample times. The results of this investigation demonstrated that immunodiffusion tests using antiserum to alkali-solubilized polyhedra can be used to detect virus infection routinely in 2nd instar and larger H. zea larvae during middle and late stages of
312
YOUNG ET AL.
disease. Since this technique is not sufficiently sensitive to detect viral infection during early stages of disease, it cannot be used to quantitate the incidence of infection within a population. This procedure shows promise, however, for utilization under field conditions for early predictions of trends in larval mortality. The advantages of this technique over more sensitive serological methods lie in its simplicity and the speed with which large samples can be tested. REFERENCES CORBETT, M. K., AND SISLER, H. D. 1964. “Plant Virology,” p. 527. University of Florida Press, Gainesville. CROWLE, A. J. 1961. Interpretation of immunodiffusion tests. Annu. Rev. Microbial. 14, 161-176. CUNNINGHAM, J. C. 1968. Serological and morphological identification of some nuclear-polyhedrosis and granulosis viruses. J. Invertebr. Pathol. 11, 132-141. DEBRE, R., AND CELERS,J. 1970. “Clinical Virology, the Evaluation and Management of Human Viral Infections,” p. 87 1. W. B. Saunders, Philadelphia, Pa.
KRYWIENCZYK,J. 1962. Immunoelectrophoretic studies of inclusion body proteins. J. Insect Pathol. 4, 185191. KRYWIENCZYK, J., AND BERGOLD, G. H. 1960a. Serological relationship of viruses from some lepidopterous and hymenopterous insects. Virology lo,3088315. KRYWIENCZYK, J., AND BERGOLD, G. H. 1960b. Serological relationship between inclusion body proteins of some Lepidoptera and Hymenoptera. J. Immunol.
84,404408.
KRYWIENCZYK, J., AND BERGOLD, G. H. 1961. Serological studies of inclusion-body proteins by agar diffusion techniques. J. fnsecf Pathol. 3, 15-24. OUCHTERLONY,0. 1962. Diffusion-in-gel methods for immunological analysis (II). Progr. AIIergy 6, 30154.
SCOTT, H. A., AND YOUNG, S. Y. 1973. Antigens associated with a nuclear polyhedrosis virus from cabbage looper larvae. J. Invertebr. Pathol. 21, 315317.
YOUNG, S. Y., AND JOHNSON, D. R. 1972. Nuclear polyhedrosis virus-specific soluble antigens in fat body of Trichoplusia ni larvae. J. Inverrebr. Pathol. 20,114-117.
YOUNG, S. Y., AND YEARIAN, W. C. 1974. Persistence of Heliothis NPV on foliage of cotton, soybean and tomato. Environ. Entomol. 3,253-255.