JOURNAL

OF INVERTEBRATE

PATHOLOGY

38, 201-208 (1981)

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

AND

P. V. VAIL

AND SUSAN

S. COLLIER

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

nicu

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.

INTRODUCTION

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.

202
MATERIALS AND

MORRIS,

VAIL,

AND

COLLIER

METHODS

Virus culture. Eggs of T. ni were obtained from either M. R. Bell at the Western Cotton Research Laboratory, Phoenix, Arizona, or from R. E. Wheeler at the Chevron Chemical Company, Richmond, California. The larvae were reared and infected with the virus isolates as described Vail et al., (1981). The small RNA virus (TRV) was purified according to the method of Morris et al. (1979) and a homogeneous virus preparation from density gradients was used for inoculum. The analysis of virus preparations by density gradient centrifugation and gel electrophoresis was as described by Morris et al. (1979). Serology. Antisera to TRV was produced in rabbits by a series of three intramuscular injections of 100-200 yg of density gradient purified virus, emulsified with Freuds adjuvant, at weekly intervals. Immunodiffusion tests were performed in plastic Petri dishes containing 0.7% Ionagar in 0.01 M phosphate-buffered saline at pH 7.4 (PBS). Larval extracts were added to wells 4 hr prior to the addition of antisera, and plates were incubated at 30C for 18 hr. The plates were washed with 0.85% NaCl, dried at 6OC, and results recorded after staining in 0.05% Coomassie blue in 50% methanol and 10% acetic acid. The enzyme-linked immunosorbent assay (ELISA) was performed according to Clark and Adams (1977) with a few moditications. Gamma globulin purification was by chromatography of crude sera (2 ml) on a 1 x 3-cm column of DEAE-Affigel Blue (Biorad Inc., Richmond, Calif.) in 0.02 M Tris, 0.28 M NaCl, 0.02% sodium azide at pH 8.0. In the initial experiments, tests were performed in microtiter plates obtained from Dynatech Laboratories and washings were performed manually between steps. Subsequent tests were done in a Gilford Model PR 50 EIA Processor utilizing specially designed cuvette strips. Plates or strips were coated with purified gamma globulin at l-5 &ml in 0.05 M

sodium carbonate, pH 9.6, for 12 hr at 4C. Antigen containing samples and alkaline phosphatase-coupled gamma globulin (1 pg/ ml) were suspended in 0.01 M PBS, pH 7.4, 0.05% Twen, 2% polyvinylpyrrolidone- 10 and 0.2% ovalbumin (PBS-T-PVP). Plates and strips were incubated at 35C on a rotary shaker at 60 rpm for 2 hr between steps, washed three times in 0.15 M NaCl, 0.1% Tween 20, and developed by the addition of 1 mg/ml p-nitrophenyl phosphate in 10% diethanolamine, pH 9.8, for 1 hr at 30C. Samples for ELISA tests were processed by homogenization of larvae or tissue samples with a mortar and pestle (usually one larva or 0.2 g of tissue) in 1 ml of 0.1 M Tris, 0.1 M NaCl, 0.02% sodium azide, pH 7.5, and 0.5 ml of carbon tetrachloride. The homogenate was clarified by centrifugation for 1- 2 min in a Beckman Microfuge B. Routinely, 0.1 ml of supernatant was removed at this stage and 0.05-ml aliquots were added to wells in microtiter plates or cuvette strips containing 0.2 ml of PBS-TPVP. Serial dilutions were performed on all samples. Virus was precipitated from the supernatant by addition of 0.2 ml of 40% PEG 6000, 1 M NaCl per milliliter of supernatant. The precipitate was collected by centrifugation and resuspended in 0.2 ml of PBS, pH 7.4, and applied to additional wells. Usually, 0.1 ml of extract was retained for electrophoretic and density gradient analysis.
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. ni larvae, then extracting and isolating the virus on sucrose density gradients and by gel electrophoresis (Morris et al., 1979). Immunodiffusion tests performed on suspensions of AcMNPV preparations and on clarified extracts of infected larvae gave inconsistent results, but satisfactory immunodiffusion results were obtained after

RNA

VIRUS

IN

AcMNPV

PREPARATIONS

203

PEG concentration of insect extracts. 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. 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. Table 1 illustrates the level of ELISA activity detected in AcMNPV preparation l-l-l which was produced in 1970. In this experiment, 20 mg of NPV formulation was suspended in 1 ml of 0.02 M PBS, 0.05% Tween 20 at pH 7.4 for 2 hr at 30C before being clarified by centrifugation and was added to antibody-coated microplate wells. A comparable, uncontaminated (as determined by bioassay) preparation of NPV (AC2) showed no reactivity. Supplementation of the NPV preparation with TRV to a concentration of 2 &ml gave comparable activity to the l-l-l preparation. The TRV content of the formulation was estimated at 1 pg/ml per 10 mg, a contamination level of 0.01%. The incidence of TRV in AcMNPV isolates has been more extensively evaluated in another paper (Vail et al., 1981).
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. ni T. rri

Absorbance at 405 nm 0.46 0.06 0.03 0.04 1.16 1.01 0.5 I 0.08 1.52 0.83
0.08 0.06

TRV-infected 7. ui' AcMNPV t I-I-lt-infected AcMNPV (ACZ)-infected Healthy T. ni

0.66 0.16 0.04 0.03

A visible, positive color reaction was evident at 0.1 OD. b NPV samples were suspended in 0.01 M PBS. pH 7.4, and clarified by centrifngation. Single larvae were extracted in 1 ml of 0.1 M Tris, 0.1 M NaCI. 0.01% sodium a&de. pH 7.4. and 0.5 ml of carbon tetrachloride. and clarified by centrifugation.

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. ni larvae and performing ELISA tests on larval extracts (Table 1). To evaluate the specificity and sensitivity of the ELISA reactivity, we fed neonate T. ni lethal (1 PIB/ mm) and sublethal doses (0.1 PIB/mm) of preparation 1-l- 1. Larvae were extracted at 12 days postinoculation and tested by both ELISA and gel electrophoresis for the presence of TRV. Individual larvae, both alive and dead, were homogenized in 1 ml of buffer and both crude and PEGconcentrated extracts were tested (Table 2). The unused portions of like samples were pooled and concentrated by ultracentrifugation at 38,000 rpm for 90 min through 1 ml of 30% sucrose in a Beckman SW 50.1 rotor. The pellets were resuspended in 0.003 M Tris-glycine, pH 8.0. and subjected to electrophoresis in 2.6% acrylamide gels (Fig. 1) and sucrose density gradient centrifugation (Fig. 2). The results of the ELISA tests showed that larvae exposed to a sublethal dosage of AcMNPV had a sporadic, lower level of infection with TRV than comparable dead insects infected with lethal dosages of NPV. All larvae exposed to the higher dosage had a higher level of detectable TRV infection (Table 2). In addition, fecal material removed from the cups of infected larvae and infected larvae themselves, contained comparable amounts of TRV. An independent estimation of the quantity of TRV was obtained by gel electrophoresis of the larval extracts (Fig. 1). The virus content was quantitated by planimetry of the gel scans as described previously (Morris et al.. 1979). The electrophoretic estimations (Table 2) directly paralleled the ELISA results confirming the identity of the serological reactions. In addition, analysis of the purified extracts by sucrose density gradient centrifugation (Fig. 2) showed that ELISA activity was

204

MORRIS,

VAIL,

AND

COLLIER

DETECTION

OF

TRV

IN

Trichnplusiu

TABLE ni LARVAE AND

2 INFECTED GEL
WITH

AcMNPV

BY ELISA,

IMMUNODIFFUSION,

ELECTROPHORESIS test IMD average fLO.5 0.08 0.15 0.85 1.12 2.5 1 0.07 1.87 test (% positive) 0 0 25 50 66 0 33 Gel electrophoresis (average &larvae)

ELISA No. of larvae tested 25 0.1 0.1 1.O 1.0 PIB/mm PIB/mm2 PIB/mm* PIB/mm* I5 8 4 20 4 4 Average wt of sample(g) 0.20 0.19 0.l* 0.2 0.2 % positive 0 33 87 100 100 0 100

Sample Control T. ni Infected T. ni (a) Alive at (b) Dead at (c) Alive at (d) Dead at Fecal pellets (a) Control (b) Infected

0 0.2 0.5 1.1 4.3 0 1.6

T. ni T. ni

(I Individual larvae extracted as in Table 1, average reactivity is for samples after concentration by PEG precipitation. b Immunodiffusion plate wells were filled with PEG-concentrated extracts, and precipitin lines were recorded after staining the plates. r Pooled samples were purified by ultracentrifugation and concentrations were estimated from planimetry of gel scans.

of

sedimentable and coincident with pure, 200 S virus. Although not illustrated, such preparations contained 35nm virions when examined in the electron microscope.

Sensitivity

of the ELISA

Test

FIG. 1. Polyacrylamide gel electrophoresis of purified TRV extracts of larvae analyzed by ELISA in Table 2. The gels were loaded with extracts equivalent to one or two infected larvae or 0.2 g of tissue and electrophoresed at 200 V for 3 hr into 2.6% gels in 0.003 M Tri-glycine, pH 8.0 (a) Control T. ni; (b) live T. ni inoculated with NPV at 0. I PIE#mm*; (c) dead T. ni inoculated at 0.1 PIB; (d) live T. ni at 1.0 PIB; (e) dead T. ni at 1.O PIB; (f) fecal extract from dead T. ni; and (g) purified TRV at 5 pg.

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. However, the purity level of the test antigen sample did affect our ability to interpret the quantitative data reliably. Figure 3 illustrates the effect of dilution of TRVcontaining extracts on ELISA activity. Each test was performed six times and the curves were constructed from the mean values. A maximum standard deviation of 0.11 was observed. All antigen samples were serially diluted fivefold in PBS-TPVP. Pure virus was serially diluted from an initial concentration of 25 pg/ml. A linear response was evident at virus concentrations ranging from 5 to 0.04 pg/rnl (Fig. 3a, b). This represented reliable detection of about 10 ng of virus. When larval extracts were analyzed after organic solvent clarification and PEG precipitation, the response curves were similar to those of pure virus (Fig. 3a). 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.

RNA

VIRUS

IN

AcMNPV

PREPARATIONS

305

I
SEDIMENTATION-

FIG. 2. Sedimentation in sucrose density gradients of purified extracts from T. ni larvae. The gradients contain (a) a healthy 7. rzi extract equivalent to two larvae (0.5 g);(b) an extract of T. ni infected with 1-1-I AcMNPV equivalent to four larvae (0.4 g); and (c) an extract of T. ni infected with TRV alone equivalent to two larvae (0.4 g). The samples were centrifuged in linear log gradients in 0.01 M Tris, pH 7.5, for 1 hr at 37,000 rpm in the SW50.1 rotor. The gradients were fractionated into 0.2-ml fractions and tested for ELISA activity (405 nm).

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. 3b). Interference was evident at the lowest dilution but the detectable ELISA reactivity was significantly less affected by dilution than pure virus. This result was consistently observed for larval extracts but was not evident in extracts of larval fras processed in a similar manner. 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. However, 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.

FIG. 3. Dilution response curves of TRV serological activity measured at 405 nm after ELBA tests. Each point represents a mean of six determinations with a maximum standard deviation of 0.1 OD. (a) The effect of dilution on: (A) pure virus at 25 pg/ml; (0) an infected larval extract after clarification and PEG purification; (A) a comparable extract of an uninfected larva; and (0) an uninfected extract supplemented with pure virus at 25 &ml. (b) The effect of dilution on: (A) pure virus at 25 &ml: (0) an infected larval extract clarified only by centrifugation; (0) an infected fras extract: and (a) an extract of uninfected larvae treated similarly.

Detection of TRV in Larval Squashes

Although quantitative detection of TRV was not satisfactory in crude extracts, direct detection in larval squashes was evaluated for routine diagnostic purposes to eliminate the more laborious purification. 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

206

MORRIS,

VAIL,

AND

COLLIER

for extraction and centrifugation. Frozen larvae were sliced into eight equal portions from the anterior to the posterior ends and crushed in the wells (Table 3). Young, newly infected larvae displayed an uneven distribution of virus, whereas TRV was detectable in all parts of older larvae as soon as 6 days postinoculation. The reliability of this approach was confirmed by indexing larvae inoculated with different doses of TRV and at different times after inoculation. We froze larvae after infection and tested in ELISA plates by squashing 2to 3-mm midbody slices (Table 4). Consistent and reliable detection of TRV was possible at 6-7 days postinoculation at all dosages tested. Detectable virus replication occurred in larvae infected at the neonate stage and in the later instars as the insects approached pupation. Incidence of Other Contaminant Viruses

California. Control T. ni reared from eggs obtained from Phoenix, however, contained 60, 90, and 125 S species. The 60 S species has been partially characterized and has many of the properties of the mini-virus group described by Longworth (1978). The other species appeared viruslike in the electron microscope (20- to 30-nm spheres), but their incidence has been too elusive to permit a more thorough study and none showed any serological relationship to TRV. Of interest is their occurrence in what appear to be healthy larvae (Fig. 4b) and the depressing effect that AcMNPV had on their synthesis (Fig. 4~). The effect was less marked by coinfection with TRV alone (Fig. 4d). These results tend to suggest that the contaminant virus problem may be somewhat ubiquitous in NPVs produced in lab-reared insects.
DISCUSSION

Throughout our investigation, after samples had been individually tested, they were routinely pooled and analyzed on sucrose density gradients. Sedimentable, viruslike species (60, 90, and 125 S) other than the 200 S species identified as TRV, were often observed in extracts of apparently healthy, as well as AcMNPV-infected, T. ni larvae. Representative density gradient profiles are illustrated in Figure 4. In this experiment, no viruslike species could be isolated from control T. ni reared from eggs obtained from the Chevron facility in Richmond,
TABLE ni LARVAE

We have demonstrated the presence of significant levels of an infectious RNA virus in preparations of AcMNPV. This NPV has been extensively studied by many investigators, yet the presence of the contaminant was discovered only recently. Since relatively large quantities of AcMNPV have been produced and used for limited field testing as a biological control agent, it becomes evident that appropriate quality control procedures designed to detect such contaminant viruses need to be developed and adopted.
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 0.02 0.05 0.41 and crushed

No. 5 A,,, 0.07 0.12 0.60 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

0.08 0.15 0.21

0.07 0.08 0.18

0.09 0.07 0.30

0.04 0.18 0.58

0.08 0.27 0.48

0.02 0.20 0.62 in the wells.

I Frozen larvae were sliced into eight equal segments Aao5 readings are an average of three larvae per test.

200 ~1 of PBS-T-PVP

RNA VIRUS IN AcMNPV TABLE

EVALUATION OF DIRECT LARVAL SQUASHES

PREPARATIONS 4
PLATES FOR THE DETECTION OF

107

IN ELISA

TRV Average A ,ir, U.08 0.68 0.79 0.80 0.79 0.06 0.12 0.47 0.62 0.04 0.07 0.57 0.79

ELISA reaction Sample (A) Dosage of TRV (i) Control (ii) Fed 25 rig/ml of TRV (iii) Fed 2.5 rig/ml (iv) Fed 0.25 rig/ml (v) Fed 0.025 &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. positive/No. tested Oil0 IO/IO IO/IO lO/lO IO/IO IO/l0 018 l/8 818 818 O/II 118 Iii.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 the concentration indicated and midbody slices were crushed directly into wells at IO days p.i, The average weight per larva was 0.02-0.03 g. Neonate larvae were fed 0.1 ml of TRV at 2.5 rig/ml and frozen at the times indicated. Whole single larvae were squashed for 3- and 6-day-old infections and 3-mm midbody slices of larvae were used for control and 9-day-old infections. Mid-development larvae (l-cm length) were fed 0. I ml of TRV at IO @ml and frozen at 8 days postinoculation. after about half had entered a prepupal stage. Midbody slices (2-3 mm) were tested.

We have compared several procedures, both physical and serological, for the detection and identification of such small non-occluded RNA viruses in insect extracts. The ELISA test proved the most effective in detecting small amounts of virus in single infected insects and NPV preparations. However, 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, requires that the contaminant viruses be identified and an antiserum be made. 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., 1979). Analyze the extract by centrifugation and electron microscopy.

(2) Isolate, characterize, and produce antisera to the viruses. (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. This approach allows rapid identification and production of contaminant-free NPV preparations (Vail et al.. 1981). The problem is, however, a complex and diffkult one to resolve and will require some extensive effort, as evidenced by our data. which suggest that several contaminant viruses may occur in at least one of the T. ni rearing systems. The problem cannot, however. be ignored if baculoviruses are to be successfully developed as alternative pesticides. The characterization of TRV led us to suspect that the virus might have some affinity for the mammalian caliciviruses (Morris et al., 1979). A serological relationship to

208

MORRIS, VAIL,

AND COLLIER and support, and B. Hillman for excellent technical assistance.

a 1

poS ,rs

REFERENCES
CLARK, M. F., AND ADAMS, A. N. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J. Gen. Virol., 34, 475-853. HESS, R. T., SUMMERS, M. D.. FALCON, L. A., AND STOLTZ, D. B. 1977. A new icosahedral insect virus: Apparent mixed nuclear infection with the baculovirus of Autographa californica. IRCS Med. Sri. 5, 562. HESS, R. T., SUMMERS, M. D., AND FALCON, L. A. 1978. A mixed virus infection in midgut cells of Autographa californica and Trichoplusia ni larvae.
J. Ultrastruct. Res., 65, 253-265.

-SEDIMENTATION

FIG. 4. Sedimentation in sucrose gradients of purified virus extracts of T. ni equivalent to four larvae each. The gradients contain (a) control larvae reared from Chevron eggs; (b) control larvae obtained from Phoenix and reared in Fresno; (c) Phoenix larvae infected with l-l-l AcMNPV in Fresno; and (d) Phoenix larvae infected with TRV in Fresno. Centrifugation conditions as in Figure 2.

known caliciviruses could not be demonstrated, but it was possible to establish that pig and human sera possessed significant levels of antibodies reactive to TRV (Morris et al., 1981). Similar data have been reported for other small RNA invertebrate viruses of this type (Longworth et al., 1973; MacCallum et al., 1979). Since detection methods exist, the prudent course of action would be to screen for and eliminate such contaminants from any baculovirus preparation being considered for field application.
ACKNOWLEDGMENTS
We thank D. E. Pinnock for assistance in the production of antiserum, D. E. Schlegel for his interest

LONGWORTH, .I. F. 1978. Small isometric viruses of invertebrates. Advun. Virus Res., 23, 103- 157. LONGWORTH, J. F., ROBERTSON. J. S., TINSLEY. T. W., ROWLANDS, D. J., AND BROWN, F. 1973. Reactions between an insect picornavirus and naturally occurring IgM antibodies in several mammalian species. Nuture (London) 242, 314-316. LONGWORTH, J. F., AND SCOTTI, P. D. 1978. Identification of nonoccluded viruses of invertebrates. In Viral Pesticides: Present Knowledge and Potential Effects on Public and Environmental Health: EPA 600/9-78-026. MACCALLUM, F., BROWN, G., AND TINSLEY, T. 1979. Antibodies in human sera reacting with an insect pathogenic virus. Intervirology. 11, 234-237. MORRIS, T. J., HESS, R. T., AND PINNOCK, D. E. 1979. Physicochemical characterization of a small RNA virus associated with baculovirus infection in Trichoplusiu ni. Intervirology:y, 11, 238-247. MORRIS, T. J., SCHLEGEL, D. E.. SOERGEL, M. E., AND SCHAFFER, F. L. 1981. Detection of naturally occurring antibodies in mammalian sera to a small RNA invertebrate virus associated with a baculovirus. Submitted for publication. SCOTTI, P. D., AND LONGWORTH, J. F. 1978. Purification of polyhedra from insect extracts contaminated with a small isometric virus. J. Invertehr. Puthol., 32, 216-218. VAIL, P. V., JAY, D. L., AND HUNTER, D. K. 1971. Cross infectivity of a nuclear polyhedrosis virus isolated from the alfalfa looper, Autogrupho culifornicu. In Proceedings IV International Colloquium on Insect Pathology, pp. 297-304. College Park, Md. VAIL, P. D., MORRIS, T. J., AND COLLIER, S. S. 1981. An RNA virus in Autogruphu culifornicu nuclear polyhedrosis virus preparations: Incidence, activity and gross pathology in T. ni. J. Invertebr. Puthol.. in press.