38, 201-208 (1981)

An RNA Virus in Autographa californica Nuclear Polyhedrosis Preparations: Detection and IdentificationlT2
Depurtment of Plant Pathology. University of Cul(forniu. Berkeley. Culiforniu 94720





Stored Producf Insects Reseurch Luhorutory. Administration, U.S. Depurtment of Agriculrure,

Agrirulturul Reseurch. Science und Educution 5578 Air Terminul Drive. Fwsno. Culiforniu 93?2?

Received September 15. 1980

A 3%nm RNA virus ofTrichop/usia ni (TRV) was detected in preparations ofAL(toyrapha cu&rnuclear polyhedrosis virus (AcMNPV). In comparisons of methods of detection of TRV (gel electrophoresis. sucrose density gradient centrifugation, and serology), the enzyme-linked immunosorbent assay (ELISA) was the most sensitive method for quantitative detection of the contaminant virus in infected larvae and AcMNPV preparations. A scheme is proposed for the detection of contaminant viruses in NPVs being developed for biological control.


The nuclear polyhedrosis virus of Autographa cafifornica (AcMNPV) is currently being evaluated as a potential microbial pest control agent because of its relatively broad host range (Vail et al., 1971). The association of this NPV with other viruses in insect-reared preparations (Hess et al., 1977, 1978) prompted our investigation of nonoccluded RNA contaminant viruses occurring in NPV preparations destined for field application. The characterization of a small RNA virus (TRV) from Trichoplusia ni infected with AcMNPV (Morris et al., 1979) and the subsequent demonstration of detectable levels of antibodies in domestic swine sera to this virus in California (Morris
t Mention of a trademark. proprietary product, or vendor does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products or vendors that may also be suitable. ? This work was supported in part by a Berkeley Campus Biomedical Research Grant and by a USDASEA/FR cooperative agreement 5%9AH2-9-478 entitled “Determination of extraneous viral contaminants in Buculovirus preparations.” 201

et al., 1981) has raised some questions about the potential biohazard that might be associated with the use of such contaminated NPV preparations. Current interest in the development of baculoviruses for insect pest control has also led to more extensive basic research on the isometric viruses of invertebrates (Longworth, 1978) and Longworth and Scotti (1978) suggested that they could become a contamination problem during baculovirus propagation. In the USA, no concerted effort has been made to adopt quality control procedures designed to detect and eliminate such contaminant viruses during the production of NPVs for pest control. However, Scotti and Longworth (1978) evaluated procedures for removal of an invertebrate enterovirus from baculovirus preparations. In this paper we have evaluated some serological and physical detection procedures to identify preparations of AcMNPV contaminated with TRV, and in a companion paper (Vail et al., 1981) we document the extent of the contamination problem and some of the biological effects of the mixed inocula.
0022-201 l/81/050201-08$01.00/0
Copyright All rights 0 1981 by Academic Press. Inc of reproduction in any form reserved.

Eggs of T. R.1 ml of supernatant was removed at this stage and 0. Richmond. Immunodiffusion tests performed on suspensions of AcMNPV preparations and on clarified extracts of infected larvae gave inconsistent results.4 (PBS). at weekly intervals. for 12 hr at 4°C.2 ml of 40% PEG 6000. AND COLLIER METHODS Virus culture. (1979) and a homogeneous virus preparation from density gradients was used for inoculum. Usually. California.1% Tween 20. The small RNA virus (TRV) was purified according to the method of Morris et al.5 ml of carbon tetrachloride. RESULTS Detection of TRV in NPV Preparations We initially detected the small RNA virus (TRV) in several preparations of AcMNPV by feeding contaminated material to susceptible T. 0. dried at 6O”C.2 ml of PBS. Bell at the Western Cotton Research Laboratory. Virus was precipitated from the supernatant by addition of 0. The enzyme-linked immunosorbent assay (ELISA) was performed according to Clark and Adams (1977) with a few moditications. Antisera to TRV was produced in rabbits by a series of three intramuscular injections of 100-200 yg of density gradient purified virus. pH 7. and 0. and results recorded after staining in 0.. Gamma globulin purification was by chromatography of crude sera (2 ml) on a 1 x 3-cm column of DEAE-Affigel Blue (Biorad Inc. VAIL. The larvae were reared and infected with the virus isolates as described Vail et al.85% NaCl.01 M phosphate-buffered saline at pH 7.) in 0. tests were performed in microtiter plates obtained from Dynatech Laboratories and washings were performed manually between steps.0.05 M sodium carbonate. Wheeler at the Chevron Chemical Company.05-ml aliquots were added to wells in microtiter plates or cuvette strips containing 0. Serology. Larval extracts were added to wells 4 hr prior to the addition of antisera. and plates were incubated at 30°C for 18 hr. The precipitate was collected by centrifugation and resuspended in 0. 1 M NaCl per milliliter of supernatant. ni were obtained from either M. 0.15 M NaCl. and applied to additional wells. pH 7. Routinely.202 MATERIALS AND MORRIS.1 M Tris. The plates were washed with 0.2 g of tissue) in 1 ml of 0. then extracting and isolating the virus on sucrose density gradients and by gel electrophoresis (Morris et al. Phoenix. In the initial experiments. pH 9.8. The homogenate was clarified by centrifugation for 1. Arizona. Richmond.6. Plates or strips were coated with purified gamma globulin at l-5 &ml in 0.7% Ionagar in 0.02% sodium azide at pH 8. (1979). 0. washed three times in 0. 2% polyvinylpyrrolidone. 0.. Serial dilutions were performed on all samples. 0.02% sodium azide. for 1 hr at 30°C. 0. 0. 0.2 ml of PBS-TPVP. emulsified with Freud’s adjuvant. Calif. but satisfactory immunodiffusion results were obtained after . and developed by the addition of 1 mg/ml p-nitrophenyl phosphate in 10% diethanolamine.5. ni larvae. pH 7.. or from R.2 min in a Beckman Microfuge B.1 ml of extract was retained for electrophoretic and density gradient analysis. The analysis of virus preparations by density gradient centrifugation and gel electrophoresis was as described by Morris et al.4.10 and 0. E. Antigen containing samples and alkaline phosphatase-coupled gamma globulin (1 pg/ ml) were suspended in 0.01 M PBS. Plates and strips were incubated at 35°C on a rotary shaker at 60 rpm for 2 hr between steps. 1979).28 M NaCl.02 M Tris. pH 9.1 M NaCl.2% ovalbumin (PBS-T-PVP).4.05% Twen. Immunodiffusion tests were performed in plastic Petri dishes containing 0. Subsequent tests were done in a Gilford Model PR 50 EIA Processor utilizing specially designed cuvette strips.05% Coomassie blue in 50% methanol and 10% acetic acid. (1981). Samples for ELISA tests were processed by homogenization of larvae or tissue samples with a mortar and pestle (usually one larva or 0.

83 0. In this experiment.16 0.. The results of the ELISA tests showed that larvae exposed to a sublethal dosage of AcMNPV had a sporadic.0.02 M PBS. and clarified by centrifugation.1 PIB/mm’) of preparation 1-l.03 0. ni larvae and performing ELISA tests on larval extracts (Table 1).46 0. we fed neonate T.. a contamination level of 0.6% acrylamide gels (Fig. ni 0. 20 mg of NPV formulation was suspended in 1 ml of 0. and subjected to electrophoresis in 2. ui' AcMNPV t I-I-lt-infected AcMNPV (ACZ)-infected Healthy T. uncontaminated (as determined by bioassay) preparation of NPV (AC2) showed no reactivity. ni T. An independent estimation of the quantity of TRV was obtained by gel electrophoresis of the larval extracts (Fig.01 0. Concentrations of TRV in NPV preparations and in extracts of young larvae that had been quickly killed by high doses of NPV were still too low for detection by this procedure.5 I 0. contained comparable amounts of TRV. Individual larvae. The pellets were resuspended in 0. Table 1 illustrates the level of ELISA activity detected in AcMNPV preparation l-l-l which was produced in 1970.003 M Tris-glycine.52 0. ni lethal (1 PIB/ mm’) and sublethal doses (0.06 TRV-infected 7.1 M NaCI. 2) showed that ELISA activity was . The TRV content of the formulation was estimated at 1 pg/ml per 10 mg.01 M PBS.04 0. 0. All larvae exposed to the higher dosage had a higher level of detectable TRV infection (Table 2).000 rpm for 90 min through 1 ml of 30% sucrose in a Beckman SW 50.1 M Tris. The incidence of TRV in AcMNPV isolates has been more extensively evaluated in another paper (Vail et al.RNA VIRUS IN AcMNPV PREPARATIONS 203 PEG concentration of insect extracts. ELISA tests proved much more sensitive for the detection of TRV and allowed for a quantitative estimation of the amount of the contaminant virus in AcMNPV preparations. 0. pH 7. The unused portions of like samples were pooled and concentrated by ultracentrifugation at 38. In addition. both alive and dead. The virus content was quantitated by planimetry of the gel scans as described previously (Morris et al.08 0. ’ Single larvae were extracted in 1 ml of 0. Detection of TRV in NPV Infected Larvae It was possible to demonstrate the infectious nature of TRV in the AcMNPV preparations by feeding them to susceptible T. and clarified by centrifngation. 1981). 1) and sucrose density gradient centrifugation (Fig.16 1.1. analysis of the purified extracts by sucrose density gradient centrifugation (Fig. 1979). 2).04 1. In addition.4. and 0. pH 7. To evaluate the specificity and sensitivity of the ELISA reactivity.1 rotor. positive color reaction was evident at 0. pH 8.05% Tween 20 at pH 7.01% sodium a&de.4 for 2 hr at 30°C before being clarified by centrifugation and was added to antibody-coated microplate wells. 0.4. fecal material removed from the cups of infected larvae and infected larvae themselves.06 0. rri Absorbance at 405 nm” 0. 1). lower level of infection with TRV than comparable dead insects infected with lethal dosages of NPV.1 OD.5 ml of carbon tetrachloride.01%. TABLE IkrEcr10~ OF AN RNA I VIRUS (TRW CONTAMINANT IN Autogruphtr POLYHEDROSIS culijiwk~r NUCLEAR VIRUS PREPARATIONS AND IN INFECTED LARVAE BY ELISA Dilution Sample l/S 11500 AcMNPV AcMNPV Pure TRV AcMNPV (1-I-l) at 20 mgiml” (AC2t at 20 mg/ml at 2 pgiml (AC21 + TRV at 2 &ml 7. Larvae were extracted at 12 days postinoculation and tested by both ELISA and gel electrophoresis for the presence of TRV.08 1.03 ” A visible. A comparable. The electrophoretic estimations (Table 2) directly paralleled the ELISA results confirming the identity of the serological reactions. Supplementation of the NPV preparation with TRV to a concentration of 2 &ml gave comparable activity to the l-l-l preparation.66 0. b NPV samples were suspended in 0. were homogenized in 1 ml of buffer and both crude and PEGconcentrated extracts were tested (Table 2).

ni at 1. (f) fecal extract from dead T. .20 0.0 PIB/mm” PIB/mm2 PIB/mm* PIB/mm* I5 8 4 20 4 4 Average wt of sample(g) 0. This represented reliable detection of about 10 ng of virus.1 0. AND COLLIER DETECTION OF TRV IN Trichnplusiu TABLE ni LARVAE AND 2 INFECTED GEL WITH AcMNPV BY ELISA. Pure virus was serially diluted from an initial concentration of 25 pg/ml.0 (a) Control T. 1.O 1. b). A linear response was evident at virus concentrations ranging from 5 to 0.O PIB. b Immunodiffusion plate wells were filled with PEG-concentrated extracts. ni Infected T.5 0. the response curves were similar to those of pure virus (Fig. The gels were loaded with extracts equivalent to one or two infected larvae or 0. A maximum standard deviation of 0.85 1. ni inoculated at 0.12 2. 3a). ELECTROPHORESIS test” IMD average fLO.5 1 0.l* 0.2 0. Each test was performed six times and the curves were constructed from the mean values.08 0. 200 S virus. ni T.6% gels in 0. Addition of virus to extracts of healthy larvae purified in the same manner confirmed that reliable quantitative estimation of virus content could be made except for some interference evident at the lowest dilution. pH 8. and precipitin lines were recorded after staining the plates. 3a.1 1. ni inoculated with NPV at 0. The ELISA test was the most sensitive of the three procedures evaluated for the detection of TRV and the most amenable to development as a routine procedure for quantitative detection of the virus. ni (I Individual larvae extracted as in Table 1. Figure 3 illustrates the effect of dilution of TRVcontaining extracts on ELISA activity.3 0 1.04 pg/rnl (Fig. IMMUNODIFFUSION. Polyacrylamide gel electrophoresis of purified TRV extracts of larvae analyzed by ELISA in Table 2. VAIL.2 % positive 0 33 87 100 100 0 100 Sample Control T. of sedimentable and coincident with pure.5 1. ni. the purity level of the test antigen sample did affect our ability to interpret the quantitative data reliably. such preparations contained 35nm virions when examined in the electron microscope. average reactivity is for samples after concentration by PEG precipitation.6 T.87 test” (% positive) 0 0 25 50 66 0 33 Gel electrophoresis’ (average &larvae) ELISA No.19 0. ni. ni (a) Alive at (b) Dead at (c) Alive at (d) Dead at Fecal pellets (a) Control (b) Infected 0 0. and (g) purified TRV at 5 pg. I PIE#mm*. (e) dead T. (c) dead T.204 MORRIS.003 M Tri-glycine.2 0. All antigen samples were serially diluted fivefold in PBS-TPVP.2 g of tissue and electrophoresed at 200 V for 3 hr into 2. r Pooled samples were purified by ultracentrifugation and concentrations were estimated from planimetry of gel scans.11 was observed.1 4.07 1. of larvae tested 25 0.15 0.0 PIB. ni at 1. (b) live T. When larval extracts were analyzed after organic solvent clarification and PEG precipitation. (d) live T. Sensitivity of the ELISA Test a b C FIG. Although not illustrated.1 PIB. However.

FIG. The gradients contain (a) a healthy 7’. Interference was evident at the lowest dilution but the detectable ELISA reactivity was significantly less affected by dilution than pure virus. The samples were centrifuged in linear log gradients in 0. (A) a comparable extract of an uninfected larva. Crushing small slices of larvae directly with disposable applicator sticks into Ab-coated wells containing 200 ~1 of PBS-T-PVP gave consistent results and eliminated the need .000 rpm in the SW50. it is evident from the results that only semiquantitative detection of the virus can be made in crude extracts even when appropriate standard curves are constructed. and (0) an uninfected extract supplemented with pure virus at 25 &ml. ni larvae. pH 7. (b) The effect of dilution on: (A) pure virus at 25 &ml: (0) an infected larval extract clarified only by centrifugation. I 5 25 EXTRACT 125 DILUTION 625 3125 Crude extracts of infected larvae not subjected to organic solvent clarification produced atypical dilution response curves (Fig.01 M Tris. 3. ni infected with 1-1-I AcMNPV equivalent to four larvae (0. (0) an infected larval extract after clarification and PEG purification.5.1 OD. 2. for 1 hr at 37. This result was consistently observed for larval extracts but was not evident in extracts of larval fras processed in a similar manner.4 g). However. Sedimentation in sucrose density gradients of purified extracts from T.(b) an extract of T.RNA VIRUS IN AcMNPV PREPARATIONS 305 I SEDIMENTATION- FIG.2-ml fractions and tested for ELISA activity (405 nm). 3b). rzi extract equivalent to two larvae (0. The gradients were fractionated into 0. Each point represents a mean of six determinations with a maximum standard deviation of 0. (a) The effect of dilution on: (A) pure virus at 25 pg/ml. Detection of TRV in Larval Squashes Although quantitative detection of TRV was not satisfactory in crude extracts. Dilution response curves of TRV serological activity measured at 405 nm after ELBA tests. direct detection in larval squashes was evaluated for routine diagnostic purposes to eliminate the more laborious purification.4 g). The shape of the crude extract curve could possibly be accounted for by a combination of protein interference at low dilutions and amplification due to the presence of soluble antigen at high dilutions.5 g). (0) an infected fras extract: and (a) an extract of uninfected larvae treated similarly. and (c) an extract of T. ni infected with TRV alone equivalent to two larvae (0.1 rotor.

The 60 S species has been partially characterized and has many of the properties of the mini-virus group described by Longworth (1978). Frozen larvae were sliced into eight equal portions from the anterior to the posterior ends and crushed in the wells (Table 3). Of interest is their occurrence in what appear to be healthy larvae (Fig. 5 A.12 0. 90. 4d). This NPV has been extensively studied by many investigators. In this experiment.21 0. however. Young.58 0..07 0.07 0. Consistent and reliable detection of TRV was possible at 6-7 days postinoculation at all dosages tested. and 125 S species. ni reared from eggs obtained from Phoenix. Sedimentable.48 0. VAIL. 4b) and the depressing effect that AcMNPV had on their synthesis (Fig. newly infected larvae displayed an uneven distribution of virus.02 0. DISCUSSION Throughout our investigation. 3 DETERMINED TEST BY CRUSHING WELLS LARVAL SLICES DISTRIBUTION OF TRV IN Trichoplusic~ DIRECTLY INTO ANTIBODY-COATED (Anterior) Sample I 2 3 Slice 4 Average 0. viruslike species (60. ni reared from eggs obtained from the Chevron facility in Richmond. but their incidence has been too elusive to permit a more thorough study and none showed any serological relationship to TRV. The other species appeared viruslike in the electron microscope (20.18 0.27 0. whereas TRV was detectable in all parts of older larvae as soon as 6 days postinoculation. Since relatively large quantities of AcMNPV have been produced and used for limited field testing as a biological control agent. Control T.” 30-nm spheres).02 0. Representative density gradient profiles are illustrated in Figure 4. We froze larvae after infection and tested in ELISA plates by squashing 2to 3-mm midbody slices (Table 4).41 and crushed No. yet the presence of the contaminant was discovered only recently.07 0. Incidence of Other Contaminant Viruses California. as well as AcMNPV-infected. 90. 4~).15 0. ni larvae. were often observed in extracts of apparently healthy. I’ Frozen larvae were sliced into eight equal segments Aao5 readings are an average of three larvae per test.18 0. no viruslike species could be isolated from control T. The effect was less marked by coinfection with TRV alone (Fig.62 in the wells.08 0. 200 ~1 of PBS-T-PVP . Detectable virus replication occurred in larvae infected at the neonate stage and in the later instars as the insects approached pupation. it becomes evident that appropriate quality control procedures designed to detect such contaminant viruses need to be developed and adopted. contained 60.. after samples had been individually tested. they were routinely pooled and analyzed on sucrose density gradients. AND COLLIER for extraction and centrifugation.04 0. TABLE ni LARVAE We have demonstrated the presence of significant levels of an infectious RNA virus in preparations of AcMNPV.09 0. These results tend to suggest that the contaminant virus problem may be somewhat ubiquitous in NPVs produced in lab-reared insects.08 0.20 0.30 0. T.05 0.08 0. and 125 S) other than the 200 S species identified as TRV.60 into 6 7 (Posterior) 8 Control larvae (3 cm) Young larvae (1 cm) at 6 days postinoculation Older larvae (3 cm) at 9 days postinoculation 0. The reliability of this approach was confirmed by indexing larvae inoculated with different doses of TRV and at different times after inoculation.206 MORRIS.

The ELISA test proved the most effective in detecting small amounts of virus in single infected insects and NPV preparations.57 0.07 0. 1979). tested Oil0 IO/IO IO/IO lO/lO IO/IO IO/l0 018 l/8 818 818 O/II 118 Iii.025 &ml (B) Time of infection” (i) Control (ii) Infected for 3 days (iii) Infected for 6 days (iv) Infected for 9 days (C) Stage of larval development” (i) Control larvae (ii) Control prepupae (iii) Infected larvae (iv) Infected prepupae No. We propose that the following scheme be adopted in screening for contaminant viruses in any NPV system being developed for biological control: (1) Extract insects infected with the NPV and process to isolate small nonoccluded viruses (Morris et al.03 g.79 0. after about half had entered a prepupal stage. a complex and diffkult one to resolve and will require some extensive effort. The average weight per larva was 0.08 0.04 0. characterize.1 ml of TRV at the concentration indicated and midbody slices were crushed directly into wells at IO days p.80 0. The problem is. I ml of TRV at IO @ml and frozen at 8 days postinoculation.62 0. We have compared several procedures.25 rig/ml (v) Fed 0. ni rearing systems.79 ELISA reaction Sample (A) Dosage of TRV” (i) Control (ii) Fed 25 rig/ml of TRV (iii) Fed 2. the adoption of this type of procedure to screen the insect colony in which the NPV is to be reared and to test the end product that is produced.5 rig/ml (iv) Fed 0.. however.RNA VIRUS IN AcMNPV TABLE EVALUATION OF DIRECT LARVAL SQUASHES PREPARATIONS 4 PLATES FOR THE DETECTION OF 107 IN ELISA TRV Average A .ir. Analyze the extract by centrifugation and electron microscopy. positive/No.79 0. both physical and serological.68 0. as evidenced by our data. U. and produce antisera to the viruses. The problem cannot. however. which suggest that several contaminant viruses may occur in at least one of the T..47 0. A serological relationship to .. 1981). Midbody slices (2-3 mm) were tested.12 0. (2) Isolate. This approach allows rapid identification and production of contaminant-free NPV preparations (Vail et al. Whole single larvae were squashed for 3.5 rig/ml and frozen at the times indicated. (3) Utilize a sensitive serological test such as ELISA to monitor for the presence of the virus in the rearing facility and during the production of the NPV.06 0. ” Mid-development larvae (l-cm length) were fed 0. However. The characterization of TRV led us to suspect that the virus might have some affinity for the mammalian caliciviruses (Morris et al. ’ Neonate larvae were fed 0. requires that the contaminant viruses be identified and an antiserum be made.i. be ignored if baculoviruses are to be successfully developed as alternative pesticides. for the detection and identification of such small non-occluded RNA viruses in insect extracts.and 6-day-old infections and 3-mm midbody slices of larvae were used for control and 9-day-old infections.5 1 Ill I % Positive 0 100 100 100 100 0 12 100 100 0 12 100 100 ” Neonate larvae were fed 0.1 ml of TRV at 2.02-0. 1979).

103.. . A mixed virus infection in midgut cells of Autographa californica and Trichoplusia ni larvae. J. Invertebr.. SUMMERS. T. 1978. V. AND HUNTER. VAIL. 1979). R. T. D. 65. 1978. 23. 11. Puthol. A new icosahedral insect virus: Apparent mixed nuclear infection with the baculovirus of Autographa californica. Md. VAIL.. J. the prudent course of action would be to screen for and eliminate such contaminants from any baculovirus preparation being considered for field application. LONGWORTH. 216-218. J. 234-237. R. 562. T. L. 475-853.. Invertehr. E. REFERENCES CLARK. AND ADAMS.” pp. Intervirology:y. Purification of polyhedra from insect extracts contaminated with a small isometric virus. F. Advun. W. J.. M. Physicochemical characterization of a small RNA virus associated with baculovirus infection in Trichoplusiu ni. (b) control larvae obtained from Phoenix and reared in Fresno. VAIL. R. T. P. MORRIS. 34. 5. 1981). B. 11. Similar data have been reported for other small RNA invertebrate viruses of this type (Longworth et al. F. a 1 ‘poS .. HESS. J. L. T. 1971. 1981. M. ROWLANDS.. F. 1973. S. M. M. J. Sri.. JAY. .. 1977. L. Since detection methods exist. ni equivalent to four larvae each. Autogrupho culifornicu. P. In “Proceedings IV International Colloquium on Insect Pathology. Virol. 238-247. F. BROWN. in press. -SEDIMENTATION FIG. F. J.. 32. Nuture (London) 242. Hillman for excellent technical assistance. L. E. LONGWORTH. HESS. G.208 MORRIS. AND SCHAFFER. A. ROBERTSON.. S.. SOERGEL. SCHLEGEL. ni. 1973. J. A. J. D. J. E. Small isometric viruses of invertebrates.. AND COLLIER. AND PINNOCK. D. T. 1978.. Centrifugation conditions as in Figure 2. Sedimentation in sucrose gradients of purified virus extracts of T. D. Reactions between an insect picornavirus and naturally occurring IgM antibodies in several mammalian species. activity and gross pathology in T. D. Detection of naturally occurring antibodies in mammalian sera to a small RNA invertebrate virus associated with a baculovirus. Submitted for publication. N. T. 314-316. Puthol. F.I. 1978.. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. P. TINSLEY. J. A. SUMMERS.. Antibodies in human sera reacting with an insect pathogenic virus. Gen. Ultrastruct. D. College Park. F. AND TINSLEY. AND LONGWORTH. Identification of nonoccluded viruses of invertebrates... MORRIS. D. K. Pinnock for assistance in the production of antiserum.. D. Cross infectivity of a nuclear polyhedrosis virus isolated from the alfalfa looper. AND SCOTTI. ACKNOWLEDGMENTS We thank D. In “Viral Pesticides: Present Knowledge and Potential Effects on Public and Environmental Health: EPA 600/9-78-026.. IRCS Med.. An RNA virus in Autogruphu culifornicu nuclear polyhedrosis virus preparations: Incidence. AND FALCON. but it was possible to establish that pig and human sera possessed significant levels of antibodies reactive to TRV (Morris et al. (c) Phoenix larvae infected with l-l-l AcMNPV in Fresno.157. 253-265. F. SCOTTI.. 4. Res. P.. Schlegel for his interest LONGWORTH.. Virus Res. 297-304. 1979. 1977. MORRIS. T. E. 1979.. D. The gradients contain (a) control larvae reared from Chevron eggs. and B. D.. S. D. J. AND COLLIER and support. FALCON. AND BROWN..... E. and (d) Phoenix larvae infected with TRV in Fresno. Intervirology. D.” MACCALLUM. 1981. known caliciviruses could not be demonstrated. MacCallum et al. AND STOLTZ..