obtained from Arthur McIntosh, Rutgers University,. Piscataway, N.J., and grown in Ham F-12 medium plus 10% FCS. All other cell lines were obtained from.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1983, p. 1085-1093 0099-2240/83/031085-09$02.00/0 Copyright © 1983, American Society for Microbiology
Vol. 45, No. 3
In Vitro Survey of Autographa californica Nuclear Polyhedrosis Virus Interaction with Nontarget Vertebrate Host Cells LOY E. VOLKMAN* AND PHYLLIS A. GOLDSMITH Department of Entomology and Parasitology, University of California, Berkeley, California 94720 Received 26 August 1982/Accepted 8 December 1982
Thirty-five nontarget host cell lines, 23 of human and 12 of nonhuman vertebrate origin, were exposed to Autographa californica nuclear polyhedrosis virus preparations derived from four different sources: polyhedra, hemolymph, cell culture medium, and cultured cells. The virus and cells were incubated together at two different temperatures, 28 or 37°C, for four different lengths of time, 16, 40, 64, or 168 h, and the cells were assayed for the presence of virus by a peroxidase-antiperoxidase detection method. The estimated sensitivity of the assay as routinely conducted was 0.98 ng of alkali-liberated viral protein and 1.95 ng of budded viral protein per mm2. No evidence of frank replication was obtained in any of the 35 cell lines tested, although virus uptake appeared to be quite common. Virus uptake was confirmed in some cases by electron microscopy. The degree of virus uptake appeared to be dependent on cell type, time and temperature of incubation, and viral phenotype. Virus purified from polyhedra was generally taken up more readily than were the other forms tested.
The nuclear polyhedrosis viruses (NPVs) of Heliothis zea, Orgyia pseudotsugata, and Lymantria dispar have been registered for use as biological pesticides by the U.S. Environmental Protection Agency. The registration of a fourth baculovirus, Autographa californica NPV (AcNPV), is pending. The responsible use of baculoviruses as pesticides requires that new information on their biological interaction with nontarget hosts continually be generated and reviewed for elucidation and evaluation of hazards that may be associated with their use (6). Accordingly, several studies have been reported in which nontarget cell lines were examined for their ability to support the replication of various NPVs. The results of these studies varied, from the observation of a frank infection of the FoghLund human amnion cell line (complete with infectious polyhedron production) by transfection with the DNA from Bombyx mori NPV (4) to the observation of no apparent interaction between alkali-liberated Heliothis NPV and four different primate cell lines (5). Intermediate between these two extremes were reports of low levels of AcNPV replication detected by the isotopic labeling of apparent progeny genomes in AcNPV-exposed Aedes aegypti (14) and Chinese hamster (12) cell lines, as well as reports of virus retention (9, 10) and uptake (2, 11) by
several other vertebrate cell lines, but with no evidence of replication. Variable factors between one study and another included not only the particular virus and cell lines used, but the form (phenotype) of the virus used to initiate infection, the conditions of incubation, the method of virus detection, and the level of sensitivity of the method used. To expand the scope of the investigations conducted to determine the possible infectivity of AcNPV in nontarget host cells and to simultaneously determine the effect of some of the above-mentioned variables, we exposed 35 nontarget cell lines, 23 of human and 12 of nonhuman vertebrate origin, to AcNPV preparations derived from four different sources: polyhedra, hemolymph, cell culture medium, and cultured cells. We incubated the virus and cells together at two different temperatures, 28 or 37°C, for four different lengths of time, 16, 40, 64, or 168 h, and assayed for virus with a peroxidase-antiperoxidase (PAP) staining method (17, 18). The sensitivity of the assay as routinely conducted was estimated to be 0.98 ng of alkali-liberated viral protein and 1.95 ng of budded viral protein per mm2. No evidence of frank replication was obtained in any of the 35 cell lines tested, although virus uptake appeared to be quite common. Virus uptake was confirmed in some cases by electron microscopy.
1085
1086
VOLKMAN AND GOLDSMITH
The form of the viral inoculum appeared to be a significant factor in the degree of uptake by some cell lines. MATERIALS AND METHODS Cell lines. The lepidopteran cell lines TN-368 and IPLB-SF-21 were maintained at 28°C in TNM-FH medium with 10% fetal calf serum (FCS) and TC-100 medium with 10% FCS, respectively, as described previously (18). The vertebrate cell lines used, their passage level, and the species, age, sex, race, and tissue of origin of the donors are listed in Tables 1 and 2. All vertebrate cell lines were maintained at 37°C in a 5% C02-95% air environment. The C3H-3W and the Hs578T cell lines were obtained from Gertrude Buehring, University of California, Berkeley. They were maintained in Dulbecco modified Eagle minimal essential medium (DMEM) and 10% FCS. The CHO-Kl cell line was obtained from Arthur McIntosh, Rutgers University, Piscataway, N.J., and grown in Ham F-12 medium plus 10% FCS. All other cell lines were obtained from Jack Weaver of the Naval Biosciences Laboratory, Oakland, Calif., and were produced with support from the National Cancer Institute, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention, under the auspices of the office of Naval Research and the regents of the University of California. These cell lines were grown and maintained in DMEM and 10% FCS. For experimental purposes, 37°C cultures grown in DMEM contained 3.7 g of NaHCO3 per liter of medium and were incubated in a 5% CO2 environment. The 28°C cultures grown in DMEM contained 0.37 g of NaHCO3 per liter of medium and were incubated in a 100% air environment. AcNPV preparations for inoculation of cell cultures. Four different types of AcNPV preparations were used to inoculate the cell cultures; the plasma membranebudded virus from cell culture (BV), intracellular virus, virus from infectious hemolymph, and virus purified from polyhedra. The AcNPV was originally supplied as cell culture-derived polyhedra by M. D. Summers, Texas A & M University, College Station, Tex., and was the cloned variant strain E2 (15). These polyhedra were used to infect laboratory-reared Trichopulsia ni larvae. Polyhedra purified from these larvae were the source of the larval-occluded, alkali-liberated virus (LOVAL) used in these studies. Virus was released from the polyhedra and gradient purified as described previously (20). Hemolymph from infected larvae was diluted in TNM-FH medium with 0.001 M dithiothreitol and used as the infectious hemolymph inoculum. Infectious hemolymph was also used to infect cultures of IPLB-SF-21 cells to generate the BV inoculum. The second generation of BV, from the culture medium of IPLB-SF-21 cells 48 h postinoculation (p.i.), was the BV used in these studies. The cells used to generate the BV were also used to generate the intracellular inoculum. After removal of the BV-containing medium, the cells were suspended in fresh medium at 106 cells per ml and homogenized for 60 s with a Tekmar Tissumizer (Tekmar Co., Cincinnati, Ohio). Large cellular fragments were pelleted by centrifugation at 1,000 x g for 15 min. The supernatant fraction was used as the intracellular virus preparation. All viral preparations except the BV were filtered
APPL. ENVIRON. MICROBIOL.
through 0.45-,um filters before use, and all were titrated on TN-368 cells. Portions of the preparations were stored frozen at -75°C until use. Routine experimental method for assessing infectivity of AcNPV in vertebrate cell lines. Monolayers of vertebrate cells were trypsinized with 0.05% trypsin and 0.02% EDTA in saline without Ca2+ or Mg2' and adjusted to 106 cells per ml in the appropriate medium. Samples (10 ,ul) of cells were spotted on 10 of 12 5-mm wells on cell culture-prepared printed glass slides. (Slides were obtained and prepared as previously described [18]). The remaining two wells per slide were spotted with 10 p.l of 104 TN-368 cells. Eight such slides were prepared for each experiment. Cells were allowed to attach for 30 to 60 min before the medium was removed and the viral inoculum was added. Each of the four virus preparations was thawed and diluted in the appropriate medium just before use. Samples (10 ,ul) of each virus preparation were added to duplicate wells and were not removed until the cells were fixed and stained. Two wells containing vertebrate cells remained virus free and served as uninoculated controls. The TN-368 cells inoculated with the LOVAL preparation served as the positive controls. The multiplicity of infection for each of the viral preparations used, as determined for TN-368 cells, was: BV, 0.24; intracellular virus, 0.4; hemolymph virus, 0.5; and LOVAL, 0.03. The slides and cells were placed in sterile petri dishes, which were in turn placed in a humidified atmosphere at either 28 or 37°C. At 16, 40, 64, and 168 h p.i., one slide from each incubation temperature group was removed, and the cells were specifically stained for the presence of AcNPV by the PAP method reported previously (17, 18), with antiserum to LOVAL at a 1:400 concentration. For some experiments, antiserum to polyhedrin was used instead. Dose-response experiments. A549 and A427 cells were prepared and spotted on slides as described above. After cell attachment, the medium was removed and duplicate wells were inoculated with 1:2 dilutions of the LOVAL preparation, starting with the routine concentration. The cells and inocula were incubated for 4 h at 28°C, the inocula were removed, and the cells were overlayed with 0.6% methylcellulose suspended in DMEM with 10% FCS. At the appropriate time, the methylcellulose was removed and the cells were stained as described above. Method of estimating assay sensitivity. The sensitivity of the PAP assay was estimated by carefully spotting 1-,ul samples of a 1:2 dilution series of known concentrations of gradient-purified LOVAL and BV onto clean glass slides. Viral protein concentrations were determined by the Bio-Rad protein assay (BioRad Laboratories, Richmond, Calif.). The drops of virus were allowed to air dry, and the residual spots were fixed and stained by the standard assay procedure (17) with antiserum to LOVAL at a 1:400 concentration. The stained spots were viewed and assessed for the presence or absence of brown reaction product. The spot with the highest virus dilution to be recognized independently by two investigators as being positive for reaction product was considered to be the endpoint of assay sensitivity. The diameter of the spot was measured, and the virus concentration per area calculated. From this value the concentration of virus necessary to produce a recognizable positive reaction
VOL. 45, 1983
Oq
o> z
o CNs 00 *- >c '-A
oo 00 *-
Z U
Z~ ~ 'IC
,Z
533
oX
*=O
0
3n 0
f cr'
+
>e
X CD _
= # H
+
rO
.0
3-
-
1087
'Ivu
£c £c
00 X
VVrr
r
C. 00 00 00 a)
11-
o.
- °o£
r
o
